U.S. patent application number 13/923274 was filed with the patent office on 2014-01-02 for nanoparticle delivery system and components thereof.
The applicant listed for this patent is Frank GU. Invention is credited to Frank GU.
Application Number | 20140005379 13/923274 |
Document ID | / |
Family ID | 49767986 |
Filed Date | 2014-01-02 |
United States Patent
Application |
20140005379 |
Kind Code |
A1 |
GU; Frank |
January 2, 2014 |
NANOPARTICLE DELIVERY SYSTEM AND COMPONENTS THEREOF
Abstract
The present disclosure relates generally to a nanoparticle
delivery system. The nanoparticles are formed from amphiphilic
block copolymers comprising polylactide and dextran. The dextran
may be conjugated to a targeting moiety in such a manner that the
surface of the nanoparticle is coated with the targeting moiety.
The size and targeting of the nanoparticles can be tuned by
controlling surface density of the targeting moiety. The present
disclosure also relates to polymers and macromolecules useful in
the preparation of the mucoadhesive nanoparticles, as well as
compositions, methods, commercial packages, kits and uses related
thereto.
Inventors: |
GU; Frank; (Waterloo,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
GU; Frank |
Waterloo |
|
CA |
|
|
Family ID: |
49767986 |
Appl. No.: |
13/923274 |
Filed: |
June 20, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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61690127 |
Jun 20, 2012 |
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Current U.S.
Class: |
536/112 |
Current CPC
Class: |
A61P 1/02 20180101; A61P
21/02 20180101; A61K 9/5161 20130101; A61P 7/12 20180101; A61P 1/10
20180101; A61P 3/04 20180101; A61P 9/08 20180101; Y02A 50/30
20180101; A61K 9/5153 20130101; A61P 25/04 20180101; A61K 47/549
20170801; A61P 11/10 20180101; A61P 1/08 20180101; A61P 9/04
20180101; A61P 31/04 20180101; A61P 5/30 20180101; A61P 9/10
20180101; A61P 25/06 20180101; A61P 35/00 20180101; A61P 37/08
20180101; Y10T 428/2982 20150115; A61P 7/10 20180101; A61P 43/00
20180101; A61K 49/0002 20130101; A61P 27/14 20180101; A61P 33/00
20180101; A61K 47/6935 20170801; A61P 11/14 20180101; A61P 15/18
20180101; A61K 9/5192 20130101; A61P 1/16 20180101; A61P 5/00
20180101; A61P 11/00 20180101; A61P 15/02 20180101; A61P 9/12
20180101; A61P 15/16 20180101; A61P 37/06 20180101; A61K 31/131
20130101; A61K 47/54 20170801; A61K 47/6937 20170801; A61K 9/006
20130101; A61P 11/02 20180101; A61P 25/28 20180101; A61P 31/00
20180101; A61K 31/69 20130101; A61P 5/38 20180101; A61P 25/18
20180101; A61P 1/06 20180101; A61P 11/04 20180101; A61P 9/00
20180101; A61K 47/6939 20170801; A61K 9/5123 20130101; A61K 45/06
20130101; A61P 3/12 20180101; A61P 15/00 20180101; A61P 25/20
20180101; A61K 31/78 20130101; A61P 19/06 20180101; A61K 38/13
20130101; A61P 17/04 20180101; A61P 25/24 20180101; A61P 27/06
20180101; A61P 25/02 20180101; A61P 25/16 20180101; A61P 27/02
20180101; A61P 27/12 20180101; A61P 31/12 20180101; A61P 33/10
20180101; A61P 13/10 20180101; A61P 29/00 20180101; A61P 3/02
20180101; A61P 3/06 20180101; A61P 3/10 20180101; A61P 7/02
20180101; A61P 31/10 20180101; A61P 27/04 20180101; A61P 31/16
20180101; A61P 35/02 20180101; A61P 1/04 20180101; A61P 11/06
20180101; A61P 25/26 20180101; A61P 31/22 20180101; A61K 47/26
20130101; A61P 9/06 20180101; A61P 19/02 20180101; A61P 1/12
20180101 |
Class at
Publication: |
536/112 |
International
Class: |
A61K 47/26 20060101
A61K047/26; A61K 31/704 20060101 A61K031/704 |
Claims
1. A block copolymer useful in the formation of a nanoparticle
delivery system for encapsulating a hydrophobic therapeutic agent,
the block copolymer having a hydrophobic portion consisting of
polylactide and a hydrophilic portion consisting of dextran, the
dextran having a plurality of functional groups capable of
conjugation to a targeting moiety.
2. The block copolymer of claim 1, which is
poly(D,L-lactide)-b-dextran.
3. The block copolymer of claim 2, wherein at least a portion of
the functional groups are conjugated to the targeting moiety.
4. The block copolymer of claim 3, wherein the targeting moiety is
phenylboronic acid.
5. A nanoparticle useful for encapsulating a hydrophobic
therapeutic agent, the nanoparticle comprising a plurality of
amphiphilic block copolymers, each copolymer having a hydrophobic
portion consisting of polylactide and a hydrophilic portion
consisting of dextran, the dextran having a plurality of functional
groups capable of conjugation to a targeting moiety.
6. The nanoparticle of claim 5, wherein the block copolymer is
poly(D,L-lactide)-b-dextran.
7. The nanoparticle of claim 6, wherein at least a portion of the
functional groups are conjugated to the targeting moiety, thereby
providing a surface-functionalized nanoparticle.
8. The nanoparticle of claim 7, wherein targeting moiety is
phenylboronic acid (PBA).
9. The nanoparticle of claim 8, wherein the size of the
nanoparticle is tunable by the surface density of phenylboronic
acid.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of priority of U.S.
Provisional Patent Application No. 61/690,127, filed Jun. 20, 2012,
which is incorporated herein by reference in its entirety.
TECHNICAL FIELD
[0002] The present disclosure relates generally to a tunable
nanoparticle delivery system. The present disclosure also relates
to components useful in the preparation of the nanoparticles, as
well as to compositions, methods, processes, commercial packages,
kits and uses related thereto.
BACKGROUND
[0003] The delivery of a drug to a patient with controlled release
of the active ingredient has been an active area of research for
decades and has been fueled by the many recent developments in
polymer science. Controlled release polymer systems can be designed
to provide a drug level in the optimum range over a longer period
of time than other drug delivery methods, thus increasing the
efficacy of the drug and minimizing problems with patient
compliance.
[0004] Nanomedicine--the fusion of nanotechnology and medicine--is
among the most promising approaches to address challenges
associated with conventional drug delivery methods. In the past
decade, drug delivery systems constructed from polymeric
nanoparticles (NPs) have been the cornerstone of progress in the
field of nanomedicine. Various types of polymeric materials have
been studied for NP drug delivery applications.
[0005] PLGA-PEG is the most widely used polymer for making
biodegradable drug delivery systems. The self-assembly of PLGA-PEG
block copolymers generally yields NPs of sizes greater than 150 nm
(Karnik, 2008). Although smaller particles can be synthesized, they
generally suffer from low drug encapsulation and rapid drug release
(Karnik, 2008). The present inventors reported that typical maximum
drug loading in PLGA-PEG was found to be 7.1 wt/wt % (Verma, 2012,
incorporated herein by reference in its entirely). Other PEG based
polymers showed drug loading ranging from 4.3 to 11.2 wt/wt %
(Shuai, 2004; He, 2010; Missirlis, 2006).
[0006] Nanoparticles have been developed as sustained release
vehicles used in the administration of small molecule drugs as well
as protein and peptide drugs and nucleic acids. The drugs are
typically encapsulated in a polymer matrix which is biodegradable
and biocompatible. As the polymer is degraded and/or as the drug
diffuses out of the polymer, the drug is released into the body.
Typically, polymers used in preparing these particles are
polyesters such as poly(lactide-co-glycolide) (PLGA), polyglycolic
acid, poly-beta-hydroxybutyrate, polyacrylic acid ester, etc. These
particles can also protect the drug from degradation by the body.
Furthermore, these particles can be administered using a wide
variety of administration routes. Various types of materials used
for synthesizing nanoparticle drug carriers have been disclosed,
for example, in US. Pat. No. 2011/0300219. Amphiphilic compound
assisted nanoparticles for targeted delivery have been disclosed,
for example, in US. Pat. No. 2010/0203142.
[0007] Targeting controlled release polymer systems (e.g., targeted
to a particular tissue or cell type or targeted to a specific
diseased tissue but not normal tissue) is desirable. It can enhance
the drug effect at the target site and reduce the amount of a drug
present in tissues of the body that are not targeted. Therefore,
with effective drug targeting, it may be possible to reduce the
amount of drug administered to treat a particular disease or
condition and undesirable side effects may also be reduced.
[0008] Various benefits can be obtained through delivery of
therapeutic agents through a mucosal tissue. For example, mucosal
delivery is generally non-invasive, thereby avoiding uncomfortable
aspects of intravenous, intramuscular, or subcutaneous delivery
means. Application of a therapeutic agent to a mucosal tissue can
also reduce the effect of first-pass metabolism and clearance by
circulating immune cells. However, given the tendency of natural
bodily fluids to clear applied therapeutic agents from the site of
administration, the administration of therapeutic agents to mucosal
sites, such as the eye, nose, mouth, stomach, intestine, rectum,
vagina, or lungs, among others, can be problematic.
[0009] Topical administration is the most common delivery method
employed for treating diseases and conditions affecting the eye,
such as corneal diseases. Common topical formulations, such as eye
drops or ointments, suffer from low ocular bioavailability due to
rapid drainage through the naso-lacrimal duct, near constant
dilution by tear turnover, and low drug permeability across the
corneal epithelium. As a result, topical formulations are normally
administered multiple times daily in order to achieve therapeutic
efficacy, resulting in a higher potential for side effects and
lower patient compliance.
[0010] Recently, formulations using NPs as drug carriers have been
proposed to overcome the limitation associated with topical
administration methods. NP carriers have been shown to improve drug
stability in water and also prolong drug activity by releasing
encapsulated compounds in a controlled manner (Ludwig, 2005;
Nagarwal 2009; Liu, 2012). NPs formulated using biodegradable
polymers, such as poly(lactic-co-glycolic acid) (PLGA), have been
tested for ocular topical drug delivery applications (Diebold,
1990; Zimmer, 1995). Poly(ethylene glycol)-based NPs have attracted
significant attention due to their ability to improve the stability
of drug carrier systems in physiological environments (Bazile,
1995; Dhar, 2008; Dong, 2007; Esmaeili, 2008).
[0011] The synthesis of surface-functionalized NP drug delivery
systems has been explored. In order to achieve mucoadhesion, the
synthesis typically requires two-stage synthesis whereby the first
stage involves the formation of NPs, while the second stage
involves the conjugation of ligands on the surface of these NPs.
Recently, a new technology demonstrated the formation of targeting
NPs using one-step synthesis whereby the formation of the NP and
the surface functionalization can be accomplished in one step (U.S.
Pat. No. 8,323,698, incorporated herein by reference). This
technology is particularly useful for applications where minimal
targeting ligand is required, e.g. for systemic bolus injections
where the number of targeting ligands on the surface must be
controlled to minimize systemic immunogenicity. When nanoparticles
are formed using the one-step method, targeting ligands may be
detected within the core of the nanoparticles. Thus, this
methodology may not be ideal where maximum targeting is
desired.
[0012] The surfaces of polymeric NPs have been functionalized with
molecular ligands that can selectively bind to the ocular mucosa to
increase precorneal drug retention (du Toit, 2011; Khutoryanskiy,
2011; Shaikh, 2011). To date, the most widely used method to
achieve mucoadhesion exploits electrostatic interactions between
the negatively charged sialic acid moieties of the corneal mucin
and cationic polymers such as chitosan (Sogias, 2008). However, the
electrostatic interactions may be hindered by various counter ions
in the tear fluid, resulting in the clearance of these NPs by tear
turnover.
[0013] A number of molecular targeting groups have been suggested
in the past for targeting the human mucosal lining: U.S. Pat. No.
7,803,392 B2 filed Dec. 8, 2011, entitled "pH-sensitive
mucoadhesive film-forming gels and wax-film composites suitable for
topical and mucosal delivery of molecules"; US Pat. 2005/0196440,
filed Sep. 8, 2005, entitled "Mucoadhesive drug delivery devices
and methods of making and using thereof"; US Pat. 2005/0281775,
filed Dec. 22, 2005, entitled "Mucoadhesive and bioadhesive
polymers"; EP 2167044 A1, filed Dec. 11, 2008, entitled
"Mucoadhesive vesicles for drug delivery"; WO 2005/117844, filed
Sep. 17, 2009, entitled "Mucoadhesive nanocomposite delivery
system"; WO 2010/096558, filed Feb. 18, 2010, entitled
"Bi-functional co-polymer use for ophthalmic and other topical and
local applications"; US Pat. 2013/0034602, filed Jul. 30, 2012,
entitled "Enteric-coated capsule containing cationic nanoparticles
for oral insulin delivery"; EP Pat. 2510930 A1, filed Apr. 15,
2011, entitled "Nanoparticles comprising half esters of poly
(methyl vinyl ether-co-maleic anhydride) and uses thereof"; U.S.
Pat. No. 8,242,165 B2, filed Oct. 26, 2007, entitled "Mucoadhesive
nanoparticles for cancer treatment"; EP Pat. 0516141 B1 filed May
29, 1992, entitled "Pharmaceutical controlled-release composition
with bioadhesive properties"; WO 1998/030207 A1, filed Jan. 14,
1998, entitled "Chitosan-gelatin a microparticles"; EP Pat. 1652517
B1, filed Jun. 17, 2004, entitled "Hyaluronic acid nanoparticles";
U.S. Pat. No. 8,361,439 B1, filed Aug. 20, 2012, entitled
"Pharmaceutical composition of nanoparticles". However, these
documents only describe mucoadhesive materials that undergo
physical interaction with the mucous lining (e.g. electrostatic
interaction between cationic chitosan materials with the negatively
charged mucin layer). The main disadvantage of physical interaction
is that it is unspecific and much weaker compared to covalent
interactions.
[0014] A few studies have reported molecular targeting groups with
potential to covalently bind to mucosal tissue. Phenylboronic acid
(PBA), which contains a phenyl substituent and two hydroxyl groups
attached to boron, has been reported to form a complex with the
diol groups of sialic acid at physiological pH (Matsumoto, 2010;
Matsumoto, 2010; Matsumoto, 2009). Another class of molecules that
can covalently bind to the mucous membrane is polymeric thiomers
(Ludwig, 2005). These thiomers are capable of forming covalent
disulfide linkage with cysteine-rich subdomains of the mucous
membrane (Khutoryanskiy, 2010). Typical examples of polymeric
thiomers include the following conjugates: poly(acrylic
acid)/cysteine (Gugg, 2004), chitosan/N-acetylcysteine (Schmitz,
2008), alginate/cysteine (Bernkop-Schnurch, 2008)
chitosan/thio-glycolic acid (Sakloetsakun, 2009) and
chitosan/thioethylamidine (Kafedjiiski, 2006). A recent study also
suggested that polymers with acrylate end groups are also capable
of binding to the thiol moieties of mucous membrane through Michael
addition (Davidovich-Pinhas and Bianco-Peled 2010). The study
demonstrated that the poly(ethylene glycol) diacrylate formed
stable covalent linkage with thiol groups of freshly extracted
porcine small intestinal mucin under physiological conditions,
which was confirmed using NMR characterization.
[0015] It is desirable to provide targeted nanoparticle delivery
systems for controlled delivery of a payload to a mucosal site. In
particular, it is desirable to provide improved mucoadhesive
delivery systems that can be retained at a mucosal site for a
sufficient period of time to provide sustained release of the
payload. It is particularly desirable to be able to tune such
delivery systems such that the extent of targeting and adhesion can
be controlled without substantially compromising the stability of
the delivery system.
SUMMARY
[0016] The present disclosure relates generally to a nanoparticle
delivery system and components thereof.
[0017] In a first aspect, there is provided a block copolymer
useful in the formation of a nanoparticle delivery system for
encapsulating a hydrophobic therapeutic agent, the block copolymer
having a hydrophobic portion consisting of polylactide and a
hydrophilic portion consisting of dextran, the dextran having a
plurality of functional groups capable of conjugation to a
targeting moiety.
[0018] In another aspect, there is nanoparticle useful for
encapsulating a hydrophobic therapeutic agent, the nanoparticle
comprising a plurality of amphiphilic block copolymers, each
copolymer having a hydrophobic portion consisting of polylactide
and a hydrophilic portion consisting of dextran, the dextran having
a plurality of functional groups capable of conjugation to a
targeting moiety.
[0019] In a further aspect, the present disclosure provides a
nanoparticle composition useful for delivery of a payload to a
mucosal site, the nanoparticle comprising a plurality of
amphiphilic macromolecules, the macromolecules comprising: a
hydrophobic portion; a hydrophilic portion comprising comprising
multiple functional moieties; and a mucosal targeting moiety,
wherein at least a portion of said functional moieties on the
hydrophilic portion are conjugated to the mucosal targeting
moiety.
[0020] In further aspect, the present disclosure provides a a
nanoparticle composition useful for delivery of a payload to a
mucosal site, the nanoparticle comprising a plurality of
amphiphilic macromolecules, the macromolecules comprising: a
hydrophobic portion comprising a biocompatible polymer selected
from a from polylactide, a polyglycolide,
poly(lactide-co-glycolide), poly(.epsilon.-caprolactone), or a
combination thereof; a hydrophilic portion comprising a
biocompatible polymer selected from polysaccharide, polynucleotide,
polypeptide, or a combination thereof, the hydrophilic portion
comprising multiple functional moieties; and a mucosal targeting
moiety selected from a phenylboronic acid (PBA) derivative, a thiol
derivative or an acrylate derivative, wherein at least a portion of
said functional moieties of the hydrophilic portion are conjugated
to the mucosal targeting moiety.
[0021] In a further embodiment, there is provided a nanoparticle
composition useful for delivery of a payload to a mucosal site, the
nanoparticle comprising a plurality of amphiphilic macromolecules,
the macromolecules each comprising: a hydrophobic biocompatible
polymer selected from a from polylactide, a polyglycolide,
poly(lactide-co-glycolide), poly(.epsilon.-caprolactone), or a
combination thereof, the hydrophobic polymer forming the core of
the nanoparticle; a hydrophilic biocompatible polymer selected from
polysaccharide, polynucleotide, polypeptide, or a combination
thereof, having multiple functional moieties, the hydrophilic
portion forming the shell of the nanoparticle; at least a portion
of the functional moieties being conjugated to a mucosal targeting
moiety selected from a phenylboronic acid (PBA) derivative, a thiol
derivative or an acrylate derivative.
[0022] In a further embodiment, there is provided a nanoparticle
composition useful for delivery of a payload to a mucosal site, the
nanoparticle comprising a plurality of amphiphilic macromolecules,
the macromolecules comprising: a hydrophobic portion comprising a
polylactide; a hydrophilic portion having multiple functional
moieties, said hydrophilic portion comprising dextran; and a
mucosal targeting moiety being a phenylboronic acid (PBA)
derivative, wherein at least a portion of said functional moieties
of the hydrophilic portion are conjugated to the mucosal targeting
moiety.
[0023] In a further embodiment, there is provided a nanoparticle
composition useful for delivery of a payload to a mucosal site, the
nanoparticle comprising a plurality of amphiphilic macromolecules,
the macromolecules each comprising a hydrophobic polylactide
polymer conjugated to a hydrophilic dextran polymer having multiple
functional moieties, at least a portion of said functional moieties
being conjugated to a phenylboronic acid (PBA) derivative.
[0024] In a further embodiment, there is provided a Dextran-p-PLA
block copolymer, wherein at least a portion of the functional
groups on the Dextran are conjugated to a targeting moiety capable
of forming a high affinity bond with a target at a mucosal
site.
[0025] In some embodiments, the nanoparticle is formed by
conjugating the polylactide to the dextran to form a nanoparticle
and subsequently surface-functionalizing the nanoparticle by
conjugating at least a portion of the functional moieties of the
dextran to the PBA derivative to achieve a desired surface density
of the PBA derivative.
[0026] In some embodiments, the nanoparticle is formed by
conjugating the polylactide to the dextran to form a nanoparticle
and subsequently reacting the functional moieties of the dextran
with PBA such that substantially all of the PBA is located in the
shell/on the surface of the nanoparticle.
[0027] In some embodiments, the core of the nanoparticle is
substantially free of targeting moiety.
[0028] In another aspect, there is provided a pharmaceutical
composition comprising a nanoparticle composition as defined in
herein, and a pharmaceutically acceptable carrier.
[0029] In another aspect, there is provided a mucoadhesive delivery
system for delivering a payload to a mucosal surface, the delivery
system comprising a nanoparticle composition as defined herein; a
pharmaceutically acceptable carrier; and a payload.
[0030] In another aspect, there is provided a method of treating or
preventing a disease or condition comprising administering to a
subject an effective amount of a nanoparticle composition or
pharmaceutical composition as described herein.
[0031] In another aspect, there is provided a use of the
nanoparticle composition or pharmaceutical composition as described
herein for treating a disease capable of being treating by
administering a therapeutic agent to a mucosal site.
[0032] In another aspect, there is provided a use of the
nanoparticle composition as described herein in the manufacture of
a medicament for treating a disease capable of being treating by
administering a therapeutic agent to a mucosal site.
[0033] In another aspect, there is provided a nanoparticle
composition or pharmaceutical composition as described herein for
use in treating a disease capable of being treating by
administering a therapeutic agent to a mucosal site.
[0034] In another aspect, there is provided a commercial package
comprising the nanoparticle composition or pharmaceutical
composition as described herein, together with instructions for use
in treating a disease.
[0035] In another aspect, there is provided a method of preparing a
nanoparticle composition useful for delivery of a payload to a
mucosal site, the method comprising: a) preparing an ampliphilic
macromolecule comprising a hydrophilic portion and a hydrophobic
portion, the hydrophilic portion comprising multiple functional
moieties; b) assembling a plurality of said macromolecules under
suitable conditions to form a nanoparticle having a hydrophobic
core and a hydrophilic shell; and c) conjugating at least a portion
of said functional moieties on the hydrophobic portion to a mucosal
targeting moiety to provide a surface-functionalized
nanoparticle.
[0036] Other aspects and features of the present disclosure will
become apparent to those ordinarily skilled in the art upon review
of the following description of specific embodiments in conjunction
with the accompanying figures.
BRIEF DESCRIPTION OF THE DRAWINGS
[0037] Embodiments of the present disclosure will now be described,
by way of example only, with reference to the attached Figures.
[0038] FIGS. 1A and B present NMR spectra at various steps of block
copolymer synthesis. FIG. 1A: Proton NMR of I. Dextran 6 kDa (D2O),
II. Dextran-NH-Et-NH-Boc (D2O), III. Dextran-NH-Et-NH2 (D2O), IV.
PLA 20 kDa (DMSO-d6), V. Dextran-Et-PLA, or PLA20-Dex6 (DMSO-d6).
FIG. 1B: Carbon NMR of block copolymer I. PLA 20 kDa, II. Dextran 6
kDa, III Dex-Et-PLA (PLA20-Dex6) confirming conjugation of Dextran
and PLA.
[0039] FIGS. 2A and B show particle size and morphology of
dextran-b-PLA NPs. FIG. 2A: Effect of MW's of PLA and Dextran on
the sizes of the NPs formed from nine different polymers using PLA
with MW 10 kDa (red), 20 kDa (green) and 50 kDa (blue), and Dextran
with MW 1.5 kDa, 6 kDa and 10 kDa. The black bars represent the
standard deviation of the particle sizes of each block copolymer.
FIG. 2B: TEM image of PLA20-Dex6 NPs (Scale bar is 100 nm) to
demonstrate spherical shape of the nanoparticles.
[0040] FIGS. 3A and B are graphs of drug encapsulation in NPs. FIG.
3A: Doxorubicin encapsulation efficiency in Dex-b-PLA and PLGA-PEG
NPs using nanoprecipitation. FIG. 3B: the corresponding drug
loading wt %. Solid gray columns are for PLA20-Dex6 NPs, solid
white columns are for PLA20-Dex10 NPs and columns with diagonal
lines pattern are for PLGA-PEG NPs (n=3; mean.+-.S.D).
[0041] FIG. 4 is a graph of in vitro Doxorubicin cumulative release
profiles from Dex-b-PLA and PLGA-PEG NPs conducted in PBS at
37.degree. C. Solid square (.box-solid.) are for PLA20-Dex10, solid
circles ( ) are for PLA20-Dex6 and solid triangles
(.tangle-solidup.) are for PLGA-PEG NPs (n=3; mean.+-.S.D).
[0042] FIG. 5 is a graph of hemolytic activity of Dex-b-PLA and
PLGA-PEG NPs for concentrations relevant to theoretical
administered dose in blood. VBS was used as a negative control and
deionized water was used as positive control in sheep erythrocytes.
Solid gray columns are for PLA20-Dex6 NPs, solid white columns are
for PLA20-Dex10 NPs and columns with diagonal lines pattern are for
PLGA-PEG NPs (n=3; mean.+-.S.D).
[0043] FIG. 6 is a graph illustrating pharmacokinetic profiles of
Dextran-b-PLA and PLGA-PEG NPs administered at 30 mg/kg i.v. to
rats. The NP concentration in blood was tracked using
[3H]-PLA-radiolabeled nanocrystals. Solid square (.box-solid.) are
for PLA20-Dex10, solid circles ( ) are for PLA20-Dex6 and solid
triangles (.tangle-solidup.) are for PLGA-PEG NPs (n=5,
mean.+-.S.D).
[0044] FIG. 7. Is a graph illustrating biodistribution of
Dextran-b-PLA and PLGA-PEG NPs in various organs in rats 24 hour
post-injection. Solid gray columns are for PLA20-Dex6 NPs, solid
white columns are for PLA20-Dex10 NPs and columns with diagonal
lines pattern are for PLGA-PEG NPs (n=5, mean.+-.S.D)**:
p<0.01.
[0045] FIG. 8 is a schematic illustration of mucoadhesion using
particulates onto ocular mucosa to circumvent the clearance
mechanisms such as tear dilution and tear turnover. Mucoadhesive
agents are present throughout the surface of the nanoparticle
carriers.
[0046] FIG. 9 is a schematic illustration of mucoadhesion of PBA
modified Dextran-b-PLA NPs onto sialic acid residues present on
ocular mucosa to circumvent the clearance mechanisms such as tear
dilution and tear turnover.
[0047] FIG. 10 is a schematic illustration of the structure of the
mucoadhesive nanoparticles with variations of targeting moieties on
the surface of the nanoparticles. The presence of multiple sites
for conjugation of targeting moiety to the surface of the
nanoparticle provides a high degree of tunability for
targeting.
[0048] FIG. 11 is a schematic illustration of one embodiment
showing the surface modified the NPs with PBA using two-step
approach: periodate oxidation of the Dextran, and conjugation of
the aldehyde groups on the oxidated Dextran with amine groups of
PBA.
[0049] FIG. 12A demonstrates .sup.1H NMR verification of the
presence of PBA on the Dex-b-PLA polymer chains. FIG. 12B
demonstrates the spherical morphology of the Dex-b-PLA_PBA NPs.
[0050] FIG. 13 demonstrates .sup.1H NMR verification of the
presence of cysteamine on the Dex-b-PLA polymer chains which would
expose thiol groups on the surface of the NPs.
[0051] FIG. 14 demonstrates the enhanced mucoadhesion property,
measured using PAS staining method, of the Dex-b-PLA NPs after
surface modified with PBA.
[0052] FIGS. 15 and 16 demonstrate the ability of the Dex-b-PLA_PBA
NPs to load up to 13.7 wt/wt % of the drug Cyclosporine A, and
their ability to release them in a sustained manner for up to 5
days in in vitro experiment.
[0053] FIG. 17 demonstrates the ability of Dex-b-PLA NPs to
encapsulate various bioactive agents. Olopatadine and Doxorubicin
were encapsulated in Dex-b-PLA NPs. Dorzolamide, Brinzolamide, and
Natamycin were encapsulated in the Dex-b-PLA_PBA NPs.
[0054] FIG. 18 demonstrates the ability of the Dex-b-PLA_PBA NPs to
release Dorzolamide (used in treatment of glaucoma) in a sustained
manner for up to 18 hours in in vitro experiment. The NPs were able
to load up to 2.8 wt/wt % Dorzolamide.
[0055] FIG. 19 demonstrates the ability of the Dex-b-PLA_PBA NPs to
release Brinzolamide (used in treatment of glaucoma) in a sustained
manner for up to 11 days in in vitro experiment. The NPs were able
to load up to 6.54 wt/wt % Brinzolamide.
[0056] FIG. 20 demonstrates the ability of the Dex-b-PLA_PBA NPs to
release Natamycin (used in treatment of ocular fungal infection) in
a sustained manner for up to 24 hours in in vitro experiment. The
NPs were able to load up to 3.88 wt/wt % Natamycin.
[0057] FIG. 21 is a schematic illustration of the partition of
nanoparticle carriers across tear fluid lipid layer.
[0058] FIG. 22 demonstrates that these various types of
nanoparticle carriers are capable of achieving high percentage
partition across the tear fluid lipid layer.
[0059] FIG. 23 demonstrates the compatibility of the exemplary NP
formulation showing no short-term toxicity effect on the ocular
surface in rabbits. NP treated and the control eyes (contralateral
eyes) after one-time administration of the NP formulation were
graded using 7 different categories (discomfort, conjunctival
redness and swelling, lid swelling, discharge, corneal
opacification, and infiltrate) by daily slit-lamp examination. The
grades demonstrate that there is no significant increase in terms
of severity of each category in the NP treated eye compared to the
control eye.
[0060] FIG. 24 shows the histopathology analysis of the cornea,
bulbar and tarsal conjunctiva, one week after the administration of
the exemplary NP formulation on rabbits. The results demonstrate
that the structure and morphology of the ocular tissues are
well-preserved after NP formulation and no sign of inflammation was
observed.
[0061] FIG. 25 demonstrates the compatibility of the exemplary NP
formulation showing no long-term toxicity effect on the ocular
surface in rabbits after weekly administration for up to 12 weeks.
Chronic response of the ocular surfaces between NP treated and the
control eyes (contralateral eyes) were evaluated similarly using 7
different categories (discomfort, conjunctival redness and
swelling, lid swelling, discharge, corneal opacification, and
infiltrate) by slit-lamp examination. The grades demonstrate that
there is no significant difference in terms of severity of each
category in the NP treated eye compared to the control eye
throughout the duration of the study.
[0062] FIG. 26 compares the chronic response of the ocular surfaces
between NP-drug treated and the control eyes (contralateral eyes)
after weekly administration of formulation containing Cyclosporine
A encapsulated NPs on rabbits. The grades of 7 different categories
(discomfort, conjunctival redness and swelling, lid swelling,
discharge, corneal opacification, and infiltrate) were obtained by
daily slit-lamp examination for up to 4 weeks. The grades
demonstrate that there is no significant difference in terms of
severity of each category in the NP treated eye compared to the
control eye throughout the duration of the study.
DETAILED DESCRIPTION
[0063] Generally, the present disclosure relates to a nanoparticle
delivery system. The nanoparticles are formed from amphiphilic
macromolecules, such as block copolymers, comprising a hydrophilic
portion and a hydrophobic portion. The hydrophobic portion
comprises multiple functional groups capable of being conjugated to
a targeting moiety, such as a mucosal targeting moiety. In an
aqueous environment, the hydrophilic portion forms the shell of the
nanoparticle providing a surface that can be functionalized by
coating the nanoparticle with a desired surface density of the
targeting moiety. The size of the nanoparticles and the surface
density of the targeting moieties can be tuned without
substantially compromising the stability of the particles. The
nanoparticles are useful for delivering a wide variety of payloads
to a mucosal site in a subject and are capable of providing
sustained release of the payload. The nanoparticles demonstrate
good loading capacity and loading efficiency.
[0064] The present disclosure also relates to components useful in
the preparation of the mucoadhesive nanoparticles, as well as
compositions, methods, processes, commercial packages, kits and
uses related thereto.
[0065] Macromolecules
[0066] The nanoparticles of the present disclosure are generally
formed by the association or assembly of amphiphilic
macromolecules. The macromolecules are composed of at least a
hydrophobic portion and at least one hydrophilic portion. The
macromolecule may comprise a hydrophobic polymer conjugated to a
hydrophilic polymer. Such macromolecules are capable of
self-assembly to form nanoparticles according to methods well known
to those skilled in the art, including nanoprecipitation
methods.
[0067] A "polymer," as used herein, refers to a molecular structure
comprising one or more repeat units (e.g. monomers), connected by
covalent bonds. The repeat units may be identical, or in some
cases, there may be more than one type of repeat unit present
within the polymer. Polymers may be obtained from natural sources
or they may be chemically synthesized. In some cases, the polymer
is a biopolymer, such as a polysaccharide, polypeptide or
polynucleotide. Biopolymers may comprise naturally-occurring
monomers or derivatives or analogs thereof, for example,
derivatives or analogs comprising modified sugars, nucleotides or
amino acids. Several such modifications are known to those skilled
in the art. In some cases, the polymer is a synthetic polymer, such
as polylactide (PLA), polyglycolide (PGA), or
poly(lactide-co-glycolide) (PLGA) or poly(.epsilon.-caprolactone)
(PCL).
[0068] If more than one type of repeat unit is present within the
polymer, then the polymer is said to be a "copolymer." The repeat
units forming a copolymer may be arranged in any fashion. For
example, the repeat units may be arranged in a random order, in an
alternating order, or in "blocks". As used herein, a "block
copolymer" comprises two or more distinct blocks or regions, e.g.
at least a first block comprising a first polymer and a second
block comprising a second polymer. It should be understood that, in
this context, the terms "first" and "second" do not describe a
particular order or number of elements but are merely descriptive.
A block copolymer may have two (a "diblock copolymer"), three (a
"triblock copolymer"), or more distinct blocks.
[0069] Block copolymers may be chemically synthesized or may be
polymeric conjugates. As used herein, a "polymeric conjugate"
describes two or more polymers that have been associated with each
other, usually by covalent bonding of two or more polymers
together. Thus, a polymeric conjugate may comprise a first polymer
and a second polymer, which have been conjugated together to form a
block copolymer where the first polymer is a first block of the
block copolymer and the second polymer is a second block of the
block copolymer. Of course, those of ordinary skill in the art will
understand that a block copolymer may, in some cases, contain
multiple blocks of polymer. For instance, a block copolymer may
comprise a first block comprising a first polymer, a second block
comprising a second polymer, and a third block comprising a third
polymer or the first polymer, etc. In addition, it should be noted
that block copolymers can also be formed, in some instances, from
other block copolymers. For example, a first block copolymer may be
conjugated to another polymer to form a new block copolymer
containing multiple types of blocks. The polymers may be conjugated
by any means known in the art and may optionally be connected by an
appropriate linker moiety.
[0070] An amphiphilic block copolymer generally has a hydrophobic
portion and a hydrophilic portion, or at least a relatively
hydrophilic portion and a relatively hydrophobic portion when two
portions are considered relative to each other. A hydrophilic
polymer is one that generally attracts water and a hydrophobic
polymer is one that generally repels water. A hydrophilic or a
hydrophobic polymer can be identified, for example, by preparing a
sample of the polymer and measuring its contact angle with water
(typically, the hydrophilic polymer will have a contact angle of
less than 60.degree., while a hydrophobic polymer will have a
contact angle of greater than about 60.degree.). In some cases, the
hydrophilicity of two or more polymers may be measured relative to
each other, i.e., a first polymer may be more hydrophilic than a
second polymer.
[0071] In some embodiments, the macromolecule is a copolymer
comprising a hydrophobic portion and a hydrophilic portion. In some
embodiments, the macromolecule is a diblock copolymer comprising a
first hydrophilic polymer and a second hydrophobic polymer. Such
configurations are generally useful for forming nanoparticles for
encapsulating hydrophobic agents of interest in an aqueous
environment, such as under physiologic conditions, since the
hydrophobic portions will shelter the hydrophobic agent in the core
region of the nanoparticle and the hydrophilic portion will form
the shell of the nanoparticle by orienting toward the aqueous
environment.
[0072] In one embodiment, the macromolecule is a Dextran-b-PLA
(Dex-b-PLA) diblock copolymer, which may optionally be
functionalized on the dextran portion with one or more targeting
moieties, such as a mucosal targeting moiety.
[0073] In some embodiments, the macromolecule is a triblock
copolymer comprising a first hydrophilic polymer, a second
hydrophobic polymer, and third hydrophilic polymer. Such
configurations are generally useful for forming nanoparticles for
encapsulating hydrophilic agents of interest in an aqueous
environment, such as under physiologic conditions.
[0074] Since the macromolecule will be exposed to bodily tissues,
it is preferable that the macromolecule comprises a biocompatible
polymer, for example, the polymer does not induce a significant
adverse response when administered to a living subject, for
example, it can be administered without causing significant
inflammation, irritation and/or acute rejection by the immune
system.
[0075] In some embodiments, the biocompatible polymer is
biodegradable, for example, the polymer is able to degrade,
chemically and/or biologically, within a physiological environment,
such as when exposed to a body tissue. For instance, the polymer
may be one that hydrolyzes spontaneously upon exposure to water
(e.g., within a subject), the polymer may degrade upon exposure to
heat (e.g., at temperatures of about 37.degree. C.). Degradation of
a polymer may occur at varying rates, depending on the polymer or
copolymer used. For example, the half-life of the polymer (the time
at which 50% of the polymer is degraded into monomers and/or other
nonpolymeric moieties) may be on the order of hours, days, weeks,
months, or years, depending on the polymer. The polymers may be
biologically degraded, e.g., by enzymatic activity or cellular
machinery, in some cases, for example, through exposure to a
lysozyme (e.g., having relatively low pH). In some cases, the
polymers may be broken down into monomers and/or other nonpolymeric
moieties that cells can either reuse or dispose of without
significant toxic effect on the cells (for example, polylactide may
be hydrolyzed to form lactic acid, polyglycolide may be hydrolyzed
to form glycolic acid, etc.).
[0076] Non-limiting examples of biodegradable polymers include, but
are not limited to, polysaccharides, polynucleotides, polypeptides,
poly(lactide) (or poly(lactic acid)), poly(glycolide) (or
poly(glycolic acid)), poly(orthoesters), poly(caprolactones),
polylysine, poly(ethylene imine), poly(acrylic acid),
poly(urethanes), poly(anhydrides), poly(esters), poly(trimethylene
carbonate), poly(ethyleneimine), poly(acrylic acid),
poly(urethane), poly(beta amino esters) or the like, and copolymers
or derivatives of these and/or other polymers, for example,
poly(lactide-co-glycolide) (PLGA).
[0077] In certain embodiments, copolymers may contain
poly(ester-ether)s, e.g., polymers having repeat units joined by
ester bonds (e.g., R--C(O)--O--R' bonds) and ether bonds (e.g.,
R--O--R' bonds). In some embodiments, the nanoparticle may further
include a polymer able to reduce immunogenicity, for example, a
poly(alkylene glycol) such as poly(ethylene glycol) ("PEG"). The
amount of PEG in the nanoparticle should be limited however, so as
not to substantiality compromise the tunability of the
nanoparticles, which is enhanced by selection of a polymer with a
backbone having multiple functional groups per monomer unit, such
as a polysaccharide, as compared to PEG which has only reactive
functional group per polymer chain. In some embodiments, the
nanoparticle composition is free of PEG.
[0078] The hydrophobic portion of the macromolecule generally
comprises a hydrophobic polymer, for example, a hydrophobic polymer
selected from polyesters, polyorthoester, polycarbonates,
polyimides, polybenzimidazoles, polyurethanes, polyureas,
polysulfides, polyethers, polysulfones, phenolic and amino
plastics, chitin and lipopolysaccharides, cholesterol,
proteoglycans, and combinations thereof. In an aqueous environment,
e.g. under physiological conditions, the hydrophobic portion will
substantially form the core of the nanoparticle.
[0079] In some embodiments, the hydrophobic portion of the
macromolecule comprises a biocompatible polymer, for example,
selected from polylactide (PLA), polyglycolide (PGA),
poly(lactide-co-glycolide) (PLGA), poly(.epsilon.-caprolactone)
(PCL), and combinations thereof. Such polymers are also
biodegradable. In a PLGA polymer, the ratios of lactide to
glycolide may be varied. In some embodiments, the hydrophobic
portion of the macromolecule comprises polylactide (PLA). In some
embodiments, the hydrophobic portion of the macromolecule comprises
polyglycolide (PGA). In some embodiments, the hydrophobic portion
of the macromolecule comprises poly(lactide-co-glycolide) (PLGA).
In some embodiments, the hydrophobic portion of the macromolecule
comprises poly(.epsilon.-caprolactone) (PCL).
[0080] In some embodiments, the hydrophobic portion is a polymer
comprising 2 or more repeat units. The hydrophilic portion may
comprise, for example, from 2 to 200,000 repeat units depending on
the size of the hydrophobic portion desired.
[0081] In some embodiments, the molecular weight of the hydrophobic
portion is in the range of about 100 g/mol to about 2,000,000
g/mol. In some embodiments, the molecular weight of the hydrophobic
portion is in the range of about 500 g/mol to about 200,000 g/mol.
In some embodiments, the molecular weight of the hydrophobic
portion is in the range of about 1,000 g/mol to about 100,000
g/mol. The unit "g/mol" in this case refers to the weight of the
hydrophobic portion per mol of the macromolecule prior to
conjugation with a targeting moiety.
[0082] In some embodiments, the molecular weight of the hydrophobic
portion is about 0.1 kDa to about 2000 kDa. In some embodiments,
the molecular weight of the hydrophobic portion is about 0.5 kDa to
about 200 kDa. In some embodiments, the molecular weight of the
hydrophobic portion is about 1 kDa to 100 kDa. These values
represent ranges prior to conjugation with a targeting moiety.
[0083] The hydrophilic portion of the macromolecule generally
comprises a polymer having multiple reactive functional groups
capable of being coupled to a targeting moiety. For example, the
polymer may comprise a backbone made up of multiple monomer units,
each monomer unit having multiple functional groups available for
conjugation to a targeting moiety. Each monomer unit may, for
example, have 2, 3, 4 or 5 functional groups. In some embodiments,
each monomer unit has 4 functional groups. The functional groups
may, for example, be independently selected from OH groups, thiol
groups, ketone groups, amine groups, and carboxylic acid groups,
among others. For example, a sugar moiety in a dextran polymer may
have 40H groups available for conjugation to a targeting moiety
(see Sheme 1).
[0084] The proportion of functional moieties conjugated to a
targeting moiety can be controlled to effect targeting. In some
embodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,
95%, 98%, 99% or 100% of the functional moieties on the surface of
the nanoparticle are conjugated to a targeting moiety.
[0085] The selection of a hydrophilic polymer having multiple
functional moieties per monomer unit allows for enhanced tunability
of the nanoparticles as compared to, for example, conventional
PEG-based nanoparticles having only one reactive functional group
at the terminal end of each PEG chain. Furthermore, in some
embodiments, the hydrophilic polymers of the present disclosure are
more hydrophilic than a PEG polymer such that the hydrophilic
portion of the macromolecule is less likely to orient toward the
core of the nanoparticle during nanoparticle formation. Since the
targeting moieties will typically be conjugated to the hydrophilic
portion, this results in a nanoparticle wherein substantially all
of the targeting moiety is on the surface of the nanoparticle) for
targeting. In such embodiments, the core of the nanoparticle is
substantially free of targeting moiety (i.e. substantially no
targeting moiety in the core of the nanoparticle.
[0086] In some embodiments, the hydrophilic portion of the
macromolecule having multiple functional groups comprises a polymer
selected from a polysaccharide, a polynucleotide, a polypeptide, or
a combination thereof. The polysaccharide, polynucleotide, or
polypeptide may be based on naturally-occurring monomers, or
derivatives or analogues thereof. Such derivatives and analogues
are known to those skilled in the art and can be readily obtained
or synthesized. In some embodiments, polysaccharides, for example,
dextran, are preferred since there are multiple functional groups
per each monomer unit.
[0087] In some embodiments, the hydrophilic portion of the
macromolecule comprises a "polysaccharide", e.g. a polymer of
monosaccharide units joined together by glycosidic linkages. Any
suitable polysaccharide may be used accordance with the present
disclosure. In some embodiments, the polysaccharide is composed of
4- to 8-carbon ring monomers, such as 5-carbon ring monomers. The
monomer rings may be heterocyclic, form example, comprising one or
more N, O or S atoms in the monomer ring. The polysaccharide may be
a "homopolysaccharide", where all of the monosaccharides in the
polysaccharide are the same type, or a "heteropolysaccharide",
where more than one type of monosaccharide is present. In some
embodiments, the polysaccharide is a "homopolysaccharide". In some
embodiments, the polysaccharide is a "heteropolysaccharide". In
some embodiments, the polysaccharide is a linear polysaccharide. In
some embodiments, the polysaccharide is a branched polysaccharide.
In some embodiments, the polysaccharide has a reducing end that can
be modified for conjugation purposes. In some embodiments, the
polysaccharide is a homopolysaccharide with a reducing end.
[0088] In some embodiments, the polysaccharide is composed of
monomers of glucose, fructose, lactose or a combination
thereof.
[0089] In some embodiments, the polysaccharide is selected from
dextran, chitosan, alginate, hyaluronic acid, heparin, chondroitin
sulphate, pectin, pullulan, amylose, cyclodextrin,
carboxymethylcellulose or a polysaccharide with thiol functional
groups conjugated to the polymer backbone.
[0090] In some embodiments, the polysaccharide is dextran,
alginate, hyaluronic acid, chitosan, cyclodextrin, or
carboxymethylcellulose. In some embodiments, the polysaccharide is
dextran.
[0091] In some embodiments, the hydrophilic portion comprises a
polynucleotide, e.g. a polymer of nucleotides. As used herein, a
"nucleotide" refers to a molecule comprising a sugar moiety, a
phosphate group, and a base (usually nitrogenous). Typically, the
nucleotide comprises one or more bases connected to a
sugar-phosphate backbone (a base connected only to a sugar moiety,
without the phosphate group, is a "nucleoside"). The sugars within
the nucleotide may be, for example, ribose sugars (a "ribonucleic
acid," or "RNA"), or deoxyribose sugars (a "deoxyribonucleic acid,"
or "DNA"). In some cases, the polymer may comprise both ribose and
deoxyribose sugars. Examples of bases include, but not limited to,
the naturally-occurring bases (e.g., adenosine or "A," thymidine or
"T," guanosine or "G," cytidine or "C," or uridine or "U"). The
nucleotide may be a naturally-occurring nucleotide or a derivative
or analog thereof. Several derivatives and analogs are known to
those skilled in the art.
[0092] In some embodiments, the hydrophilic portion comprises a
polypeptide, e.g. a polymer of amino acids. The amino acid may be a
naturally-occurring amino acid or a derivative or analog thereof.
Several derivatives and analogs are known to those skilled in the
art. In some embodiments, at least a portion (e.g. greater than
50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 100%) of the nucleotides in
the polynucleotide have side chains with reactive functional groups
capable of being conjugated to the targeting moiety.
[0093] In some embodiments, the hydrophilic portion is a polymer
comprising 2 or more repeat units. The hydrophilic portion may
comprise, for example, 2 to 100,000 repeat units depending on
desired size of the nanoparticle.
[0094] In some embodiments, the molecular weight of the hydrophilic
portion ranges from about 100 g/mol to about 1,000,000 g/mol. In
some embodiments, the molecular weight of the hydrophilic portion
ranges from about 500 g/mol to 100,000 g/mol. In some embodiments,
the molecular weight of the hydrophilic portion ranges from about
1,000 g/mol to about 50,000 g/mol. The unit "g/mol" in this case
refers to the weight of the hydrophilic portion per mol of the
macromolecule prior to conjugation with a targeting moiety.
[0095] In some embodiments, the molecular weight of the hydrophilic
portion ranges from about 0.1 kDa to about 1,000 kDa. In some
embodiments, the molecular weight of the hydrophilic portion ranges
from about 0.5 kDa to 100 kDa. In some embodiments, the molecular
weight of the hydrophilic portion ranges from about 1 kDa to 50
kDa. These values represent ranges prior to conjugation with a
targeting moiety.
[0096] The relative amount of hydrophobic polymer to hydrophilic
polymer in the macromolecule may be any suitable ratio that
provides the desired characteristics of the resulting nanoparticle.
In some embodiments, the molecular weight of the hydrophobic
portion is larger than the molecular weight of the hydrophilic
portion. In some embodiments, the molecular weight of the
hydrophilic portion is larger than the molecular weight of the
hydrophobic portion.
[0097] In some embodiments, the ratio of the molecular weight of
the hydrophobic portion to the hydrophilic portion (hydrophobic
portion:hydrophilic portion) is a about 0.1:1 to 100:1. In some
embodiments, the molecular weight ratio is about 0.5:1 to about
50:1. In some embodiments, the molecular weight ratio is about 1:1
to about 10:1. In some embodiments, the molecular weight ratio is
about 1:1 to about 5:1, about 1:1 to about 4:1, about 1:1 to about
3:1, or about 1:1 to about 2:1. In some embodiments, the molecular
weight ratio is about 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4 or
1:5. These values prepresent ratios before conjugation of the
targeting moiety. A skilled person will be albe to determine a
suitable ratio based on the particular polymers selected and the
agent of interest to be encapsulated.
[0098] Targeting
[0099] The macromolecules described herein are conjugated to a
targeting moiety, such that the targeting moiety located on the
surface of the nanoparticle when the nanoparticle is formed to
thereby surface-functionalize nanoparticle. The interaction between
the targeting moiety and a target at the mucosal site directs the
nanoparticle to a particular site and/or increases the retention
time of the nanoparticle at a particular site compared to a
nanoparticle with no targeting moiety. Any suitable targeting
moiety may be selected. Examples of targeting moieties include, but
are not limited to, small molecules, polynucleotides, polypeptides,
polysaccharides, fatty acids, lipids, and antibodies.
[0100] The targeting moiety may be a mucosal targeting moiety. As
used herein, a "mucosal targeting moiety" is a targeting moiety
capable of binding to a target expressed at the mucosal site. In
some embodiments, the nanoparticles may comprise more than one type
of mucosal targeting moiety. For example, an individual
macromolecule may be functionalized with two or more different
targeting moieties, or the nanoparticle may be formed from two or
more macromolecules, each being functionalized with a different
targeting moiety.
[0101] The term "binding," as used herein, refers to the
interaction between a corresponding pair of molecules or portions
thereof that exhibit mutual affinity or binding capacity, typically
due to specific or non-specific binding or interaction, including,
but not limited to, biochemical, physiological, electrostatic
and/or chemical interactions. In some cases, the targeting moiety
is able to selectively bind to a target expressed at the mucosal
site, for example, a molecule, receptor or residue expressed at the
mucosal site. "Selective binding", as used herein, refers to a
targeting moiety, which may be a small molecule or a large
molecule, that is able to preferentially bind to or recognize a
particular target or subset of targets, to a substantially higher
degree than to others. The target may, for example, be a biological
substrate that is preferentially expressed at the musosal site,
such as mucin or a receptor or a glycoprotein or a polysaccharide
or residue expressed on the surface of an epithelial cell. In some
cases, the binding is a high affinity binding the binding, such as
covalent bonding, van der Waal force or hydrogen bonding.
Preferably, the binding is covalent binding. For example, in some
cases, the target may possess functional groups reactive with the
targeting moiety and in a particular configuration that permits
covalent binding of the targeting moiety.
[0102] In some embodiments, the targeting moiety is capable of
binding to carbohydrate residues that contain cis-diol groups, for
example, galactose, N-acetylgalactosamine, N-acetyl-glucosamine,
fucose, and sialic acids. Such carbohydrate residues may, for
example, be present on mucin. In some embodiments, the carbohydrate
residue is a sialic acid residue. In some embodiments, the
targeting moiety is a boronic acid derivative capable of binding a
cis-diol group on a sialic acid residue. In some embodiments, the
targeting moiety is a phenylboronic acid (PBA) derivative.
[0103] In some embodiments, the targeting moiety is a thiol
derivative or an acrylate derivative capable of binding to thiol
groups of cysteine moieties. Cysteine moieties may, for example, be
present on mucin. In some embodiments, the targeting moiety is a
thiol derivative, such as cysteamine. In some embodiments, the
targeting molecule may be cysteamine derivative capable of forming
a disulfide linkage with a cysteine moiety on the mucous membrane.
In some embodiments, the targeting moiety is an acrylate derivative
capable of binding to hydroxyl groups of the glycoproteins on the
mucous membrane. Acrylate derivatives include, but are not limited
to methacrylate, ethyl acrylate, and diacrylate. In some
embodiments, the targeting moiety is an acrylate derivative
selected from methacrylate, ethyl acrylate, and diacrylate.
[0104] In some embodiments, the targeting moiety is a phenylboronic
acid (PBA) derivative, a thol derivative or an acrylate
derivative.
[0105] In some embodiments, the targeting moiety is the hydrophobic
portion of the macromolecule comprises PLA; the hydrophilic portion
comprises dextran; and the targeting moiety comprises PBA. In some
embodiments, the targeting moiety is the hydrophobic portion is
PLA; the hydrophilic portion is dextran, and the targeting moiety
is PBA.
[0106] In some embodiments, the targeting moiety is a biological
moiety. Non-limiting examples of biological moieties include a
peptide, a protein, an enzyme, a nucleic acid, a fatty acid, a
hormone, an antibody, a carbohydrate, a peptidoglycan, a
glycopeptide, or the like. In some cases, the biological moiety may
be relatively large, for example, for peptides, nucleic acids, or
the like. For example, the biological moiety may have a molecular
weight of at least about 1,000 Da, at least about 2,500 Da, at
least about 3000 Da, at least about 4000 Da, or at least about
5,000 Da, etc. Relatively large targeting moieties may be useful,
in some cases, for differentiating between cells. For instance, in
some cases, smaller targeting moieties (e.g., less than about 1000
Da) may not have adequate specificity for certain targeting
applications, such as mucosal targeting applications. In contrast,
larger molecular weight targeting moieties can offer a much higher
targeting affinity and/or specificity. For example, a targeting
moiety may offer smaller dissociation constants, e.g., tighter
binding. However, in other embodiments, the targeting moiety may be
relatively small, for example, having a molecular weight of less
than about 1,000 Da or less than about 500 Da.
[0107] Nanoparticles
[0108] Another aspect of the disclosure is directed to
nanoparticles formed generally from the association of
macromolecules, such as the macromolecules described above. The
nanoparticles demonstrated effective targeting and adhesion, as
well as sustained release of payload at the mucosal site.
[0109] Under appropriate conditions, the macromolecules are capable
of assembling to form a nanoparticle of the core-shell type, where
the core of the nanoparticle is relatively hydrophobic in
comparison to the shell. Alternatively, under different conditions,
the core of the nanoparticle may be relatively hydrophilic in
comparison to the shell. The shell provides a surface of the
nanoparticles, which may comprise a targeting moiety at a desired
surface density, such that the nanoparticles are coated with the
targeting moiety.
[0110] The nanoparticles may have a substantially spherical shape
(i.e., the particles generally appear to be spherical). Such
nanoparticles may also be referred to as "nanospheres" or
"nanovesicles" due to their generally spherical shape and the
formation of a cavity within the nanoparticle. It will be
understood that the particles, for example, upon swelling or
shrinkage, may adopt a non-spherical configuration.
[0111] The nanoparticles formed have an average particle size of
less than about 1000 nm (1 micrometer). In some embodiments, the
average particle size is less than about 500 nm, less than about
300 nm, less than about 200 nm, less than about 150 nm, less than
about 100 nm, less than about 75 nm, less than about 60 nm, less
than about 50 nm, less than about 30 nm, less than about 10 nm,
less than about 3 nm, or less than about 1 nm in some cases. In
some cases, particles less than 150 nm are preferred, for example,
such particles are better able to penetrate the tear layer of the
eye compared to larger particles.
[0112] In some embodiments, the average particle size is between
about 0.1 nm and about 1000 nm, about 1 nm and about 500 nm, about
1 nm and about 300 nm, about 1 nm and about 200 nm, about 1 nm and
about 150 nm, about 1 nm and about 100 nm, about 1 nm and about 50
nm, about 10 nm and about 150 nm, about 10 nm and about 100 nm,
about 10 nm and about 75 nm, about 10 nm and about 60 nm, and about
10 nm and about 50 nm, or about 20 and about 40 nm.
[0113] As used herein, "particle size" refers to the average
characteristic dimension of a population of nanoparticles formed,
where the characteristic dimension of a particle is the diameter of
a perfect sphere having the same volume as the particle. A
population of nanoparticles may, for example, include at least 20
particles, at least 50 particles, at least 100 particles, at least
300 particles, at least 1,000 particles, at least 3,000 particles,
at least 5,000 particles, at least 10,000 particles, or at least
50,000 particles. Various embodiments of the present invention are
directed to such populations of particles.
[0114] In some embodiments, the particles may each be substantially
the same shape and/or size, in which case the population is
"monodisperse". For example, the particles may have a distribution
of particle sizes such that no more than about 5% or about 10% of
the particles have a particle size greater than about 10% greater
than the average particle size of the particles, and in some cases,
such that no more than about 8%, about 5%, about 3%, about 1%,
about 0.3%, about 0.1%, about 0.03%, or about 0.01% have a particle
size greater than about 10% greater than the average particle size
of the particles. In some cases, no more than about 5% of the
particles have a particle size greater than about 5%, about 3%,
about 1%, about 0.3%, about 0.1%, about 0.03%, or about 0.01%
greater than the average particle size of the particles.
[0115] In some embodiments, the particles have an interior core and
an exterior shell which forms the surface of the nanoparticle,
where the shell has a composition different from the core i.e.,
there may be at least one polymer or moiety present in shell but
not in the core (or vice versa), and/or at least one polymer or
moiety present in the core and/or the shell at differing
concentrations.
[0116] In some cases, the core of the particle is more hydrophobic
than the shell of the particle. In some cases, a drug or other
payload may be hydrophobic, and therefore readily associates with
the relatively hydrophobic interior of the particle. The drug or
other payload may thus be contained within the interior of the
particle, which may thus shelter it from the external environment
surrounding the particle (or vice versa). A targeting moiety
present on the surface of the particle may allow the particle to
become localized at a particular targeting site, for instance, a
mucosal site. The drug or other payload may then, in some cases, be
released from the particle and allowed to interact with the
particular targeting site.
[0117] Yet another aspect of the disclosure is directed to
nanoparticles having more than one polymer or macromolecule
present. For example, in some embodiments, particles may contain
more than one distinguishable macromolecule, and the ratios of the
two (or more) macromolecules may be independently controlled, which
allows for the control of properties of the particle. For instance,
a first macromolecule may be a biocompatible polymeric conjugate,
such as a block copolymer, comprising a targeting moiety, and a
second macromolecule may comprise a biocompatible polymer but no
targeting moiety, or the second macromolecule may contain a
distinguishable biocompatible polymer from the first macromolecule.
Control of the amounts of these macromolecules within the polymeric
particle may thus be used to control various physical, biological,
or chemical properties of the particle, for instance, the size of
the particle (e.g., by varying the molecular weights of one or both
polymers), the surface charge (e.g., by controlling the ratios of
the polymers if the polymers have different charges or terminal
groups), the surface hydrophilicity (e.g., if the polymers have
different molecular weights and/or hydrophilicities), the surface
density of the targeting moiety (e.g., by controlling the ratios of
the two or more polymers), etc.
[0118] Tunable Nanoparticles
[0119] The nanoparticles described herein are highly tunable.
[0120] For example, the size of the nanoparticles can be tuned by
adjusting the molecular weight and/or composition of the
hydrophobic portion and/or the hydrophilic portion. The particular
targeting moiety selected, as well as the surface density of the
targeting moiety on the surface of the nanoparticles, will also
impact the particle size.
[0121] It should be noted that increasing size of the hydrophilic
and/or hydrophobic polymer components does not always result in a
larger particle size. For example, in some cases, longer polymer
chains may be more flexible and capable of folding to produce a
more compact particle whereas a shorter polymer chain may be
confined to a more linear configuration. Selection of a branched
verus a linear polymer can also impact particle size. A skilled
person will be able to select suitable polymers for a particular
application.
[0122] The hydrophilic portion of the macromolecules will form the
shell of the nanoparticles in an aqueous environment. The
hydrophilic portion is selected such that it has multiple
functional moieties for conjugation to a targeting moiety. The
proportion of functional moieties conjugated to a targeting moiety
can be controlled, at least in part, by the amount of targeting
moiety added to the conjugation reaction. In general, the more
functional moieties present on the hydrophilic portion, the higher
the degree of tunability of the nanoparticles. In general, the
nanoparticles disclosed herein are thus more tunable than PEG-based
nanoparticles having only one functional moiety per PEG chain, or
other similar polymers.
[0123] By adjusting the surface density of the targeting moiety,
the extent of targeting can be controlled. A high surface density
of the targeting moiety can be achieved with the nanoparticles
disclosed herein due to the presence of multiple functional
moieties on the hydrophilic portion. Advantageously, a skilled
person will be able to control the extent of targeting without
substantially compromising the stability of the nanoparticle
delivery system. In some embodiments, an optimal density can be
determined in which maximum targeting is achieved without
substantially compromising the stability of the nanoparticle.
[0124] In some embodiments, a majority (e.g. at least 50%, 60%,
70%, 80%, 90%, 95%, 98%, 99%, or 100%) of the mucosal targeting
moieties are located on the surface of the nanoparticle. It is
understood that, in some cases, a portion of the mucosal targeting
moieties may be located within the core of the nanoparticle when
the nanoparticle forms, depending on the components of the
nanoparticle and the method utilized. For instance, where a
one-step method is used, it is possible that some of the targeting
moieties may orient toward the core of the particles.
[0125] The selection of a hydrophilic polymer having multiple
functional moieties along the polymer backbone renders the
hydrophilic portion of the molecule more hydrophilic than other
polymers, such as PEG, thus the hydrophilic portion is more likely
to orient toward an aqueous environment. Since the targeting moiety
is conjugated to the hydrophilic portion, typically after formation
of a nanoparticle, substantially all of the targeting moieties are
located on the surface of the nanoparticle as opposed to the
core.
[0126] In some embodiments, substantially all (e.g. at least 95%,
96%, 97%.sub., 98%.sub., 99%, or 100%) of the mucosal targeting
moieties are located on the surface of the nanoparticle. Localizing
substantially all of the targeting moieties to the surface of the
nanoparticles enhances targeting efficiency. The selection of a
hydrophilic polymer having multiple functional moieties along the
polymer backbone renders the hydrophilic portion of the molecule
more hydrophilic than other polymers, such as PEG having only one
functional moiety, thus the hydrophilic portion is more likely to
orient toward an aqueous environment. Since the targeting moiety is
conjugated to the hydrophilic portion, typically after formation of
a nanoparticle, substantially all of the targeting moieties are
located on the surface of the nanoparticle as opposed to the
core.
[0127] The nanoparticles can be tuned by controlling the surface
density of the targeting moieties on the nanoparticle. A skilled
person will be able to precisely tune the nanoparticle for a
particular application without substantially compromising the
stability of the nanoparticles.
[0128] In some embodiments, the surface density of the targeting
moiety is about 1 per nm.sup.2 to 15 per nm.sup.2, about 1 per
nm.sup.2 to 10 per nm.sup.2, about 1 per nm.sup.2 to 5 per
nm.sup.2, about 1 per nm.sup.2 to about 15 per nm.sup.2, about 3
per nm.sup.2 to about 12 per nm.sup.2, or from about 5 per nm.sup.2
to about 10 per nm.sup.2.
[0129] In some embodiments, the surface density of the targeting
moiety is about 1, 2, 3, 4, 5, 6, 7, 8, 8, 9, 11, 12, 13, 14 or 15
per nm.sup.2.
[0130] In some embodiments, the nanoparticle is approximately 10 nm
in size and the density of targeting moieties on the surface of the
nanoparticle (i.e. surface density) ranges from about 50 to about
3,500, from about 500 to about 3500, or from about 1000 to about
3500 per nanoparticle.
[0131] In some embodiments, the nanoparticle is approximately 30 nm
in size and the density of targeting moieties on the surface of the
nanoparticle (i.e. surface density) ranges from about 50 to about
30000, from about 1000 to about 30000, or from about 10000 to about
30000 per nanoparticle.
[0132] In some embodiments, the nanoparticle is approximately 50 nm
in size and the density of targeting moieties on the surface of the
nanoparticle (i.e. surface density) ranges from about 50 to about
90000, from about 3000 to about 90000, or from about 30000 to about
90000 per nanoparticle.
[0133] In some embodiments, the nanoparticle is approximately 100
nm in size and the density of targeting moieties on the surface of
the nanoparticle (i.e. surface density) ranges from about 50 to
about 350000, from about 10000 to about 350000, or from about
100000 to about 350000 per nanoparticle.
[0134] In some embodiments, the nanoparticle is approximately 150
nm in size and the density of targeting moieties on the surface of
the nanoparticle (i.e. surface density) ranges from about 50 to
about 800000, from about 30000 to about 800000, or from about
300000 to about 800000 per nanoparticle.
[0135] In some embodiments, the nanoparticle is approximately 200
nm in size and the density of targeting moieties on the surface of
the nanoparticle (i.e. surface density) ranges from about 50 to
about 1,500,000, from about 60000 to about 1,500,000, or from about
600000 to about 1,500,000 per nanoparticle.
[0136] In some embodiments, the nanoparticle is approximately 250
nm in size and the density of targeting moieties on the surface of
the nanoparticle (i.e. surface density) ranges from about 50 to
about 2,500,000, from about 100,000 to about 2,500,000, or from
about 1,000,000 to about 2,500,000 per nanoparticle.
[0137] In some embodiments, the nanoparticle is approximately 300
nm in size and the density of targeting moieties on the surface of
the nanoparticle (i.e. surface density) ranges from about 50 to
about 3,500,000, from about 150,000 to about 3,500,000, or from
about 1,500,000 to about 3,500,000 per nanoparticle.
[0138] In some embodiments, the surface density of the targeting
moiety tunable by the amount of targeting moiety added in the
reaction during the functionalization step(s).
[0139] In some embodiments, the density of a phenylboronic acid
derivative on the nanoparticle surface is tuneable by the amount of
phenylboronic acid added in the reaction to control the extent of
mucoadhesion properties of the nanoparticles.
[0140] In some embodiments, the density of a cysteamine derivative
on the nanoparticle surface is tuneable by the amount of cysteamine
derivative added in the reaction to control the extent of
mucoadhesion properties of the nanoparticles.
[0141] In some embodiments, the density of an acrylate derivative
on the nanoparticle surface is tuneable by the amount of acrylate
derivative added in the reaction to control the extent of
mucoadhesion properties of the nanoparticles.
[0142] The optimal density of surface functional groups may be
determined by those skilled in the art in order to achieve balance
between the extent of mucoadhesion and the colloidal stability of
the nanoparticles.
[0143] In some embodiments, the nanoparticles are dispersed in
aqueous medium. The aqueous medium may, for example, be a
physiologically compatible aqueous medium.
[0144] Controlled Release
[0145] A controlled release system, as used herein, refers to a
nanoparticle delivery system capable of delivering a payload, such
as a therapeutic agent, a diagnostic agent, a prognostic, a
prophylactic agent, to a body tissue, such as a mucous membrane,
where the payload is released in a predesigned or controlled
manner. For example, the active agent may be released in a constant
manner over a predetermined period of time, the active agent may be
released in a cyclic manner over a predetermined period of time, or
an environmental condition or external event may trigger the
release of the active agent. The controlled release polymer system
may include a polymer that is biocompatible, and in some cases, the
polymer is biodegradable. In some cases, the nanoparticles
disclosed herein are part of a controlled release delivery system.
The nanoparticles disclosed herein demonstrated sustained release
of payload.
[0146] The mucosal targeting moiety assists in retaining the
nanoparticles at the mucosal site, i.e. for a longer time that the
same nanoparticle without the targeting moiety, such that
controlled delivery of a payload at the mucosal site can be
achieved. The controlled delivery may include sustained
delivery.
[0147] In some embodiments, in the payload is released from the
nanoparticle for a sustained period of at least 24, 36, 48, 60, 72,
84, of 96 hours. In some embodiments, the payload is released from
the nanoparticle for a sustained period of at least 1, 2, 3, 4, 5,
6, 7 or 8 days. In some embodiments, the payload is released is
released from the nanoparticle for a sustained period of at least 1
week. In some embodiments, the payload is released is released from
the nanoparticle for a sustained period of at least 1 month.
[0148] In some embodiments at least 50% of the payload is released
within the first 24 hours. In other embodiments, at least 10% the
payload is released within the first 6 hours.
[0149] Payload
[0150] A wide variety of payloads can be loaded into the
nanoparticles described herein. As used herein, the "payload" may
be any agent of interest to be delivered to a mucosal site, for
example, a therapeutic agent (e.g. drug), a diagnostic agent, a
prophylactic agent, an imaging agent, or a combination thereof. In
some embodiments, the payload is a single agent of interest. In
other embodiments, the payload comprises more than one agent of
interest, for example, a combination of two or more agents of
interest. In some embodiments, the payload comprises 2, 3 or 4
agents of interest. For example, the payload may comprise two or
more agents of interest selected from a therapeutic agent, a
diagnostic agent, a prophylactic agent, an imaging agent and
combinations thereof.
[0151] When combined with a payload, the nanoparticles described
herein are useful as a mucoadhesive nanoparticle delivery system
for delivering the payload to a mucosal site. In some embodiments,
the payload is predominantly encapsulated within the core of the
nanoparticle. By "predominantly" it is meant that more than 60%,
70%, 80%, 90%, 95% or 99% of the payload is encapsulated within the
core of the nanoparticle. It will be understood that, depending on
the composition of the nanoparticle and the payload, a portion of
the payload could also be distributed within the shell of the
nanoparticle and/or on the surface of the nanoparticle.
[0152] In some embodiments, the payload comprises a hydrophobic
agent. For example, the payload may be a hydrophobic therapeutic
agent, a hydrophobic diagnostic agent, a hydrophobic prophylactic
agent or a hydrophobic imaging agent. In one embodiment, the
payload is a hydrophobic therapeutic agent. In one embodiment, the
payload is a hydrophobic diagnostic agent. In one embodiment, the
payload is a hydrophobic prophylactic agent. In one embodiment, the
payload is a hydrophobic imaging agent. The encapsulation of
hydrophobic compounds in the nanoparticles is due to the
hydrophobic interaction between the hydrophobic agent and the
hydrophobic portions of the copolymers that form the core of the
nanoparticles.
[0153] In some embodiments, the payload comprises a hydrophilic
agent. For example, the payload may be a hydrophilic therapeutic
agent, a hydrophilic diagnostic agent, a hydrophilic prophylactic
agent or a hydrophilic imaging agent. In one embodiment, the
payload is a hydrophilic therapeutic agent. In one embodiment, the
payload is a hydrophilic diagnostic agent. In one embodiment, the
payload is a hydrophilic prophylactic agent. In one embodiment, the
payload is a hydrophilic imaging agent. It will be understood that
the composition of the nanoparticle would be modified to
encapsulate a hydrophilic payload, for example, a triblock
copolymer comprising a first hydrophilic block, and second
hydrophobic block and a third hydrophilic block could be used. Such
modifications are well known to those skilled in the art.
[0154] The nanoparticles described herein were found to have good
loading capacity and efficiency. The loading capacity of various
drugs using exemplary Dex-b-PLA (optionally surface functionalized
with PBA) nanoparticles is demonstrated in the Examples. In some
embodiments, the nanoparticles disclosed herein have higher loading
capacity than reported for conventional PEG-based polymers.
Naturally, loading capacity will be affected by the composition of
the nanoparticles and the choice of payload.
[0155] In some embodiments, the loading capacity is in the range of
about 1 to about 40% wt/wt, about 1 to about 30% wt/wt., about 1 to
about 20%, 1 to about 10%, about 1% to about 8%, about 1% to about
6%, about 1% to about 5%, about 1% to about 3%, or about 1% to
about 2%. The loading capacity (%) is calculated here as the
molecular weight of encapsulated drug over the entire weight of the
nanoparticle multiplied by 100. The total weight of the
nanoparticle refers to the weight of the nanoparticle including the
targeting moiety and the encapsulated drug.
[0156] In some embodiments, the loading capacity is up to about
40%, up to about 30% wt/wt., up to about 20%, up to about 10%, up
to about 8%, up to about 6%, up to about 5%, up to about 3%, up to
about 2%, or up to about 1%.
[0157] In some embodiments the payload has a molecular weight of
about 0.001 kDa to 100 kDa, about 0.01 kDa to 50 kDa, about 0.1 kDa
to 10 kDa.
[0158] In some embodiments, the payload has a diameter of about
0.01 nm to about 300 nm, about 0.01 nm to about 100 nm, about 0.01
nm to about 50 nm.
[0159] Non-limiting examples of potentially suitable therapeutic
agents include antimicrobial agents, analgesics, antinflammatory
agents, IOP lowering agents, counterirritants, coagulation
modifying agents, diuretics, sympathomimetics, anorexics, antacids
and other gastrointestinal agents; antiparasitics, antidepressants,
antihypertensives, anticholinergics, stimulants, antihormones,
central and respiratory stimulants, drug antagonists,
lipid-regulating agents, uricosurics, cardiac glycosides,
electrolytes, ergot and derivatives thereof, expectorants,
hypnotics and sedatives, antidiabetic agents, dopaminergic agents,
antiemetics, muscle relaxants, para-sympathomimetics,
anticonvulsants, antihistamines, beta-blockers, purgatives,
antiarrhythmics, contrast materials, radiopharmaceuticals,
antiallergic agents, tranquilizers, vasodilators, antiviral agents,
and antineoplastic or cytostatic agents or other agents with
anticancer properties, or a combination thereof. Other suitable
therapeutic agents may be selected from contraceptives and vitamins
as well as micro- and macronutrients. Still other examples include
antiinfectives such as antibiotics and antiviral agents; analgesics
and analgesic combinations; anorexics; antiheimintics;
antiarthritics; antiasthmatic agents; anticonvulsants;
antidepressants; antidiuretic agents; antidiarrleals;
antihistamines; antiinflammatory agents; antimigraine preparations;
antinauseants; antineoplastics; antiparkinsonism drugs;
antipruritics; antipsychotics; antipyretics, antispasmodics;
anticholinergics; sympathomimetics; xanthine derivatives;
cardiovascular preparations including calcium channel blockers and
beta-blockers such as pindolol and antiarrhythmics;
antihypertensives; diuretics; vasodilators including general
coronary, peripheral and cerebral; central nervous system
stimulants; cough and cold preparations, including decongestants;
hormones such as estradiol and other steroids, including
corticosteroids; hypnotics; immunosuppressives; muscle relaxants;
parasympatholytics; psychostimulants; sedatives; and tranquilizers;
and naturally derived or genetically engineered proteins,
polysaccharides, glycoproteins, or lipoproteins.
[0160] Further non-limiting examples of drugs include timolol,
betaxolol, metipranolol, dorzolamide, brinzolamide, neptazane,
acetazolamide, alphagan, xalatan, bimatoprost, travaprost,
olopatadine, ketotifen, acyclovir, gancyclovir, valcyclovir,
doxorubicin, mitomycin, cisplatin, daunorubicin, bleomycin,
actinomycin D, neocarzinostatin, carboplatin, stratoplatin, Ara-C.
Other examples include Capoten, Monopril, Pravachol, Avapro,
Plavix, Cefzil, Duricef/Ultracef, Azactam, Videx, Zerit, Maxipime,
VePesid, Paraplatin, Platinol, Taxol, UFT, Buspar, Serzone, Stadol
NS, Estrace, Glucophage (Bristol-Myers Squibb); Ceclor, Lorabid,
Dynabac, Prozac, Darvon, Permax, Zyprexa, Humalog, Axid, Gemzar,
Evista (Eli Lily); Vasotec/Vaseretic, Mevacor, Zocor,
Prinivil/Prinizide, Plendil, Cozaar/Hyzaar, Pepcid, Prilosec,
Primaxin, Noroxin, Recombivax HB, Varivax, Timoptic/XE, Trusopt,
Proscar, Fosamax, Sinemet, Crixivan, Propecia, Vioxx, Singulair,
Maxalt, Ivermectin (Merck & Co.); Diflucan, Unasyn, Sulperazon,
Zithromax, Trovan, Procardia XL, Cardura, Norvasc, Dofetilide,
Feldene, Zoloft, Zeldox, Glucotrol XL, Zyrtec, Eletriptan, Viagra,
Droloxifene, Aricept, Lipitor (Pfizer); Vantin, Rescriptor,
Vistide, Genotropin, Micronase/Glyn./Glyb., Fragmin, Total Medrol,
Xanax/alprazolam, Sermion, Halcion/triazolam, Freedox, Dostinex,
Edronax, Mirapex, Pharmorubicin, Adriamycin, Camptosar, Remisar,
Depo-Provera, Caverject, Detrusitol, Estring, Healon, Xalatan,
Rogaine (Pharmacia & Upjohn); Lopid, Accrupil, Dilantin,
Cognex, Neurontin, Loestrin, Dilzem, Fempatch, Estrostep, Rezulin,
Lipitor, Omnicef, FemHRT, Suramin, or Clinafloxacin (Warner
Lambert).
[0161] Further non-limiting examples of therapeutic agents that can
be included within a particle of the present invention include
acebutolol, acetaminophen, acetohydroxamic acid, acetophenazine,
acyclovir, adrenocorticoids, allopurinol, alprazolam, aluminum
hydroxide, amantadine, ambenonium, amiloride, aminobenzoate
potassium, amobarbital, amoxicillin, amphetamine, ampicillin,
androgens, anesthetics, anticoagulants, anticonvulsants-dione type,
antithyroid medicine, appetite suppressants, aspirin, atenolol,
atropine, azatadine, bacampicillin, baclofen, beclomethasone,
belladonna, bendroflumethiazide, benzoyl peroxide, benzthiazide,
benztropine, betamethasone, betha nechol, biperiden, bisacodyl,
bromocriptine, bromodiphenhydramine, brompheniramine, buclizine,
bumetanide, busulfan, butabarbital, butaperazine, caffeine, calcium
carbonate, captopril, carbamazepine, carbenicillin, carbidopa &
levodopa, carbinoxamine inhibitors, carbonic anhydsase,
carisoprodol, carphenazine, cascara, cefaclor, cefadroxil,
cephalexin, cephradine, chlophedianol, chloral hydrate,
chlorambucil, chloramphenicol, chlordiazepoxide, chloroquine,
chlorothiazide, chlorotrianisene, chlorpheniramine, 6.times.
chlorpromazine, chlorpropamide, chlorprothixene, chlorthalidone,
chlorzoxazone, cholestyramine, cimetidine, cinoxacin, clemastine,
clidinium, clindamycin, clofibrate, clomiphere, clonidine,
clorazepate, cloxacillin, colochicine, coloestipol, conjugated
estrogen, contraceptives, cortisone, cromolyn, cyclacillin,
cyclandelate, cyclizine, cyclobenzaprine, cyclophosphamide,
cyclothiazide, cycrimine, cyproheptadine, danazol, danthron,
dantrolene, dapsone, dextroamphetamine, dexamethasone,
dexchlorpheniramine, dextromethorphan, diazepan, dicloxacillin,
dicyclomine, diethylstilbestrol, diflunisal, digitalis, diltiazen,
dimenhydrinate, dimethindene, diphenhydramine, diphenidol,
diphenoxylate & atrophive, diphenylopyraline, dipyradamole,
disopyramide, disulfuram, divalporex, docusate calcium, docusate
potassium, docusate sodium, doxyloamine, dronabinol ephedrine,
epinephrine, ergoloidmesylates, ergonovine, ergotamine,
erythromycins, esterified estrogens, estradiol, estrogen, estrone,
estropipute, etharynic acid, ethchlorvynol, ethinyl estradiol,
ethopropazine, ethosaximide, ethotoin, fenoprofen, ferrous
fumarate, ferrous gluconate, ferrous sulfate, flavoxate, flecamide,
fluphenazine, fluprednisolone, flurazepam, folic acid, furosemide,
gemfibrozil, glipizide, glyburide, glycopyrrolate, gold compounds,
griseofiwin, guaifenesin, guanabenz, guanadrel, guanethidine,
halazepam, haloperidol, hetacillin, hexobarbital, hydralazine,
hydrochlorothiazide, hydrocortisone (cortisol), hydroflunethiazide,
hydroxychloroquine, hydroxyzine, hyoscyamine, ibuprofen,
indapamide, indomethacin, insulin, iofoquinol, iron-polysaccharide,
isoetharine, isoniazid, isopropamide isoproterenol, isotretinoin,
isoxsuprine, kaolin & pectin, ketoconazole, lactulose,
levodopa, lincomycin liothyronine, liotrix, lithium, loperamide,
lorazepam, magnesium hydroxide, magnesium sulfate, magnesium
trisilicate, maprotiline, meclizine, meclofenamate,
medroxyproyesterone, melenamic acid, melphalan, mephenyloin,
mephobarbital, meprobamate, mercaptopurine, mesoridazine,
metaproterenol, metaxalone, methamphetamine, methaqualone,
metharbital, methenamine, methicillin, methocarbamol, methotrexate,
methsuximide, methyclothinzide, methylcellulose, methyldopa,
methylergonovine, methylphenidate, methylprednisolone,
methysergide, metoclopramide, matolazone, metoprolol,
metronidazole, minoxidil, mitotane, monamine oxidase inhibitors,
nadolol, nafcillin, nalidixic acid, naproxen, narcotic analgesics,
neomycin, neostigmine, niacin, nicotine, nifedipine, nitrates,
nitrofurantoin, nomifensine, norethindrone, norethindrone acetate,
norgestrel, nylidrin, nystafin, orphenadrine, oxacillin, oxazepam,
oxprenolol, oxymetazoline, oxyphenbutazone, pancrelipase,
pantothenic acid, papaverine, para-aminosalicylic acid,
paramethasone, paregoric, pemoline, penicillamine, penicillin,
penicillin-v, pentobarbital, perphenazine, phenacetin,
phenazopyridine, pheniramine, phenobarbital, phenolphthalein,
phenprocoumon, phensuximide, phenylbutazone, phenylephrine,
phenylpropanolamine, phenyl toloxamine, phenyloin, pilocarpine,
pindolol, piper acetazine, piroxicam, poloxamer, polycarbophil
calcium, polythiazide, potassium supplements, pruzepam, prazosin,
prednisolone, prednisone, primidone, probenecid, probucol,
procainamide, procarbazine, prochlorperazine, procyclidine,
promazine, promethazine, propantheline, propranolol,
pseudoephedrine, psoralens, syllium, pyridostigmine, pyrodoxine,
pyrilamine, pyrvinium, quinestrol, quinethazone, uinidine, quinine,
ranitidine, rauwolfia alkaloids, riboflavin, rifampin, ritodrine,
alicylates, scopolamine, secobarbital, senna, sannosides a & b,
simethicone, sodium bicarbonate, sodium phosphate, sodium fluoride,
spironolactone, sucrulfate, sulfacytine, sulfamethoxazole,
sulfasalazine, sulfinpyrazone, sulfisoxazole, sulindac, talbutal,
tamazepam, terbutaline, terfenadine, terphinhydrate, teracyclines,
thiabendazole, thiamine, thioridazine, thiothixene, thyroblobulin,
thyroid, thyroxine, ticarcillin, timolol, tocamide, tolazamide,
tolbutamide, tolmetin trozodone, tretinoin, triamcinolone,
trianterene, triazolam, trichlormethiazide, tricyclic
antidepressants, tridhexethyl, trifluoperazine, triflupromazine,
trihexyphenidyl, trimeprazine, trimethobenzamine, trimethoprim,
tripclennamine, triprolidine, valproic acid, verapamil, vitamin A,
vitamin B12, vitamin C, vitamin D, vitamin E, vitamin K, xanthine,
and the like.
[0162] As another example, if the targeting moiety targets a cancer
cell, then the payload may be an anti-cancer drug such as
20-epi-1,25 dihydroxyvitamin D3,4-ipomeanol, 5-ethynyluracil,
9-dihydrotaxol, abiraterone, acivicin, aclarubicin, acodazole
hydrochloride, acronine, acylfulvene, adecypenol, adozelesin,
aldesleukin, all-tk antagonists, altretamine, ambamustine,
ambomycin, ametantrone acetate, amidox, amifostine,
aminoglutethimide, aminolevulinic acid, amrubicin, amsacrine,
anagrelide, anastrozole, andrographolide, angiogenesis inhibitors,
antagonist D, antagonist G, antarelix, anthramycin,
anti-dorsalizing morphogenetic protein-1, antiestrogen,
antineoplaston, antisense oligonucleotides, aphidicolin glycinate,
apoptosis gene modulators, apoptosis regulators, apurinic acid,
ARA-CDP-DL-PTBA, arginine deaminase, asparaginase, asperlin,
asulacrine, atamestane, atrimustine, axinastatin 1, axinastatin 2,
axinastatin 3, azacitidine, azasetron, azatoxin, azatyrosine,
azetepa, azotomycin, baccatin III derivatives, balanol, batimastat,
benzochlorins, benzodepa, benzoylstaurosporine, beta lactam
derivatives, beta-alethine, betaclamycin B, betulinic acid, BFGF
inhibitor, bicalutamide, bisantrene, bisantrene hydrochloride,
bisaziridinylspermine, bisnafide, bisnafide dimesylate, bistratene
A, bizelesin, bleomycin, bleomycin sulfate, BRC/ABL antagonists,
breflate, brequinar sodium, bropirimine, budotitane, busulfan,
buthionine sulfoximine, cactinomycin, calcipotriol, calphostin C,
calusterone, camptothecin derivatives, canarypox IL-2,
capecitabine, caracemide, carbetimer, carboplatin,
carboxamide-amino-triazole, carboxyamidotriazole, carest M3,
carmustine, carn 700, cartilage derived inhibitor, carubicin
hydrochloride, carzelesin, casein kinase inhibitors,
castanospermine, cecropin B, cedefingol, cetrorelix, chlorambucil,
chlorins, chloroquinoxaline sulfonamide, cicaprost, cirolemycin,
cisplatin, cis-porphyrin, cladribine, clomifene analogs,
clotrimazole, collismycin A, collismycin B, combretastatin A4,
combretastatin analog, conagenin, crambescidin 816, crisnatol,
crisnatol mesylate, cryptophycin 8, cryptophycin A derivatives,
curacin A, cyclopentanthraquinones, cyclophosphamide, cycloplatam,
cypemycin, cytarabine, cytarabine ocfosfate, cytolytic factor,
cytostatin, dacarbazine, dacliximab, dactinomycin, daunorubicin
hydrochloride, decitabine, dehydrodidemnin B, deslorelin,
dexifosfamide, dexormaplatin, dexrazoxane, dexverapamil,
dezaguanine, dezaguanine mesylate, diaziquone, didemnin B, didox,
diethylnorspermine, dihydro-5-azacytidine, dioxamycin, diphenyl
spiromustine, docetaxel, docosanol, dolasetron, doxifluridine,
doxorubicin, doxorubicin hydrochloride, droloxifene, droloxifene
citrate, dromostanolone propionate, dronabinol, duazomycin,
duocarmycin SA, ebselen, ecomustine, edatrexate, edelfosine,
edrecolomab, eflornithine, eflornithine hydrochloride, elemene,
elsamitrucin, emitefur, enloplatin, enpromate, epipropidine,
epirubicin, epirubicin hydrochloride, epristeride, erbulozole,
erythrocyte gene therapy vector system, esorubicin hydrochloride,
estramustine, estramustine analog, estramustine phosphate sodium,
estrogen agonists, estrogen antagonists, etanidazole, etoposide,
etoposide phosphate, etoprine, exemestane, fadrozole, fadrozole
hydrochloride, fazarabine, fenretinide, filgrastim, finasteride,
flavopiridol, flezelastine, floxuridine, fluasterone, fludarabine,
fludarabine phosphate, fluorodaunorunicin hydrochloride,
fluorouracil, fluorocitabine, forfenimex, formestane, fosquidone,
fostriecin, fostriecin sodium, fotemustine, gadolinium texaphyrin,
gallium nitrate, galocitabine, ganirelix, gelatinase inhibitors,
gemcitabine, gemcitabine hydrochloride, glutathione inhibitors,
hepsulfam, heregulin, hexamethylene bisacetamide, hydroxyurea,
hypericin, ibandronic acid, idarubicin, idarubicin hydrochloride,
idoxifene, idramantone, ifosfamide, ilmofosine, ilomastat,
imidazoacridones, imiquimod, immunostimulant peptides, insulin-like
growth factor-1 receptor inhibitor, interferon agonists, interferon
alpha-2A, interferon alpha-2B, interferon alpha-N1, interferon
alpha-N3, interferon beta-IA, interferon gamma-IB, interferons,
interleukins, iobenguane, iododoxorubicin, iproplatin, irinotecan,
irinotecan hydrochloride, iroplact, irsogladine, isobengazole,
isohomohalicondrin B, itasetron, jasplakinolide, kahalalide F,
lamellarin-N triacetate, lanreotide, lanreotide acetate,
leinamycin, lenograstim, lentinan sulfate, leptolstatin, letrozole,
leukemia inhibiting factor, leukocyte alpha interferon, leuprolide
acetate, leuprolide/estrogen/progesterone, leuprorelin, levamisole,
liarozole, liarozole hydrochloride, linear polyamine analog,
lipophilic disaccharide peptide, lipophilic platinum compounds,
lissoclinamide 7, lobaplatin, lombricine, lometrexol, lometrexol
sodium, lomustine, lonidamine, losoxantrone, losoxantrone
hydrochloride, lovastatin, loxoribine, lurtotecan, lutetium
texaphyrin, lysofylline, lytic peptides, maitansine, mannostatin A,
marimastat, masoprocol, maspin, matrilysin inhibitors, matrix
metalloproteinase inhibitors, maytansine, mechlorethamine
hydrochloride, megestrol acetate, melengestrol acetate, melphalan,
menogaril, merbarone, mercaptopurine, meterelin, methioninase,
methotrexate, methotrexate sodium, metoclopramide, metoprine,
meturedepa, microalgal protein kinase C inhibitors, MIF inhibitor,
mifepristone, miltefosine, mirimostim, mismatched double stranded
RNA, mitindomide, mitocarcin, mitocromin, mitogillin, mitoguazone,
mitolactol, mitomalcin, mitomycin, mitomycin analogs, mitonafide,
mitosper, mitotane, mitotoxin fibroblast growth factor-saporin,
mitoxantrone, mitoxantrone hydrochloride, mofarotene, molgramostim,
monoclonal antibody, human chorionic gonadotrophin, monophosphoryl
lipid a/myobacterium cell wall SK, mopidamol, multiple drug
resistance gene inhibitor, multiple tumor suppressor 1-based
therapy, mustard anticancer agent, mycaperoxide B, mycobacterial
cell wall extract, mycophenolic acid, myriaporone,
n-acetyldinaline, nafarelin, nagrestip, naloxone/pentazocine,
napavin, naphterpin, nartograstim, nedaplatin, nemorubicin,
neridronic acid, neutral endopeptidase, nilutamide, nisamycin,
nitric oxide modulators, nitroxide antioxidant, nitrullyn,
nocodazole, nogalamycin, n-substituted benzamides,
06-benzylguanine, octreotide, okicenone, oligonucleotides,
onapristone, ondansetron, oracin, oral cytokine inducer,
ormaplatin, osaterone, oxaliplatin, oxaunomycin, oxisuran,
paclitaxel, paclitaxel analogs, paclitaxel derivatives, palauamine,
palmitoylrhizoxin, pamidronic acid, panaxytriol, panomifene,
parabactin, pazelliptine, pegaspargase, peldesine, peliomycin,
pentamustine, pentosan polysulfate sodium, pentostatin, pentrozole,
peplomycin sulfate, perflubron, perfosfamide, perillyl alcohol,
phenazinomycin, phenylacetate, phosphatase inhibitors, picibanil,
pilocarpine hydrochloride, pipobroman, piposulfan, pirarubicin,
piritrexim, piroxantrone hydrochloride, placetin A, placetin B,
plasminogen activator inhibitor, platinum complex, platinum
compounds, platinum-triamine complex, plicamycin, plomestane,
porfimer sodium, porfiromycin, prednimustine, procarbazine
hydrochloride, propyl bis-acridone, prostaglandin J2, prostatic
carcinoma antiandrogen, proteasome inhibitors, protein A-based
immune modulator, protein kinase C inhibitor, protein tyrosine
phosphatase inhibitors, purine nucleoside phosphorylase inhibitors,
puromycin, puromycin hydrochloride, purpurins, pyrazofurin,
pyrazoloacridine, pyridoxylated hemoglobin polyoxyethylene
conjugate, RAF antagonists, raltitrexed, ramosetron, RAS farnesyl
protein transferase inhibitors, RAS inhibitors, RAS-GAP inhibitor,
retelliptine demethylated, rhenium RE 186 etidronate, rhizoxin,
riboprine; ribozymes, RII retinamide, RNAi, rogletimide,
rohitukine, romurtide, roquinimex, rubiginone B1, ruboxyl,
safingol, safingol hydrochloride, saintopin, sarcnu, sarcophytol A,
sargramostim, SDI 1 mimetics, semustine, senescence derived
inhibitor 1, sense oligonucleotides, signal transduction
inhibitors, signal transduction modulators, simtrazene, single
chain antigen binding protein, sizofuran, sobuzoxane, sodium
borocaptate, sodium phenylacetate, solverol, somatomedin binding
protein, sonermin, sparfosate sodium, sparfosic acid, sparsomycin,
spicamycin D, spirogermanium hydrochloride, spiromustine,
spiroplatin, splenopentin, spongistatin 1, squalamine, stem cell
inhibitor, stem-cell division inhibitors, stipiamide,
streptonigrin, streptozocin, stromelysin inhibitors, sulfinosine,
sulofenur, superactive vasoactive intestinal peptide antagonist,
suradista, suramin, swainsonine, synthetic glycosaminoglycans,
talisomycin, tallimustine, tamoxifen methiodide, tauromustine,
tazarotene, tecogalan sodium, tegafur, tellurapyrylium, telomerase
inhibitors, teloxantrone hydrochloride, temoporfin, temozolomide,
teniposide, teroxirone, testolactone, tetrachlorodecaoxide,
tetrazomine, thaliblastine, thalidomide, thiamiprine, thiocoraline,
thioguanine, thiotepa, thrombopoietin, thrombopoietin mimetic,
thymalfasin, thymopoietin receptor agonist, thymotrinan, thyroid
stimulating hormone, tiazofurin, tin ethyl etiopurpurin,
tirapazamine, titanocene dichloride, topotecan hydrochloride,
topsentin, toremifene, toremifene citrate, totipotent stem cell
factor, translation inhibitors, trestolone acetate, tretinoin,
triacetyluridine, triciribine, triciribine phosphate, trimetrexate,
trimetrexate glucuronate, triptorelin, tropisetron, tubulozole
hydrochloride, turosteride, tyrosine kinase inhibitors,
tyrphostins, UBC inhibitors, ubenimex, uracil mustard, uredepa,
urogenital sinus-derived growth inhibitory factor, urokinase
receptor antagonists, vapreotide, variolin B, velaresol, veramine,
verdins, verteporfin, vinblastine sulfate, vincristine sulfate,
vindesine, vindesine sulfate, vinepidine sulfate, vinglycinate
sulfate, vinleurosine sulfate, vinorelbine, vinorelbine tartrate,
vinrosidine sulfate, vinxaltine, vinzolidine sulfate, vitaxin,
vorozole, zanoterone, zeniplatin, zilascorb, zinostatin, zinostatin
stimalamer, or zorubicin hydrochloride. In one embodiment, the
therapeutic agent is doxorubicin.
[0163] In some embodiments, the therapeutic agent is an agent used
for treating or preventing a disease or condition that affects the
eye (e.g. an ophthalmic agent). Non-limiting examples of ophthalmic
agents include lubricants, demulcents, antibiotics, antivirals
(e.g. acyclovir, gancyclovir, valcyclovir), antiallergic agents
(e.g. antihistamine, e.g. olopatadine), IOP lowering agents,
counterirritants, acetazolamide, alphagan, antazoline, aspirin,
atropine, azelastine, bacitracin, betaxolol, bimatoprost, botanical
drugs including zeaxanthine lutein, lycopene brimonodine,
brinzolamide, carbachol, carteolol, ciprofloxacin, ofloxacin,
cromalyn, cyclosporine (including cyclosporine pro-drugs and
cyclosporine derivatives), other immunomodulators, dapiprazole,
dexamethasone, diclofenac, dipivifren, dorzolamide, epinephrine,
erythromycin, fluoromethalone, flurbiprofen, gentamycin, glaucoma
medications (e.g. prostaglandins, carbonic anhydrase inhibitors,
epinephrine or alpha-agonists, beta-blockers), gramicidin,
homatropine, hydrocortisone, hyoscine, keterolac, ibuprofen,
ketotifen, latanaprost, levobunolol, levocabastine, levofloxin,
lotepprednol, medrysone, methazolamide, metipranolol, naphazoline,
natamycin, nedocromil, neomycin, neptazane, neuroprotective agents,
nonsteroidal anti-inflammatory agents, nepafanec, norfloxacin,
ofloxacin, olopatadine, oxymetazoline, pemirolast, pheniramine,
phenylephrine, pilocarpine, povidone, prednisolone, proparacaine,
scopolamine, tetracaine, steroids, sulfacetamide, tetrahydrozoline,
hypertonic tears, timolal, tobramycin, travaprost, trifluridine,
trimethiprim, tropicamide, unoprostone, xalatan, and zinc. Prodrugs
and related compounds, as well as new active pharmaceutical
ingredients can be used with the delivery system described
herein.
[0164] In one embodiment, the therapeutic agent is an ophthalmic
agent selected from cycloprorin A, timolol, betaxolol,
metipranolol, dorzolamide, brinzolamide, natamycin, neptazane,
acetazolamide, alphagan, xalatan, bimatoprost, travaprost,
olopatadine, ketotifen, acyclovir, gancyclovir, valcyclovir. In one
embodiment, the therapeutic agent is cyclosporine A, natamycin,
olopatadine, brinzolamide or dorzolamine.
[0165] In one embodiment, the therapeutic agent is an ophthalmic
agent used to treat glaucoma, such as an agent used to reduce a
sign and/or symptom of glaucoma, for example, and agent used to
reduce intraocular pressure associated with ocular hypertension. In
some embodiments, the therapeutic agent is a glaucoma medication,
such as a prostaglandin, carbonic anhydrase inhibitor, epinephrine
or alpha-agonist, or a beta-blocker. In some embodiments, the
therapeutic agent is Dorzolamide, Brinzolamide, Brimonidine,
timolol, or latanoprost.
[0166] In one embodiment, the therapeutic agent is an ophthalmic
agent used to treat allergic conjunctivitis, such as an agent used
to reduce a sign and/or symptom of allergic conjunctivitis. In one
embodiment, the therapeutic agent is olopatadine.
[0167] In one embodiment, the therapeutic agent is an ophthalmic
agent used to treat keratoconjunctivitis sicca (KCS) or "dry eye",
such as an agent used to reduce a sign and/or symptom of KCS. In
one embodiment, the therapeutic agent is cyclosporine A.
[0168] In one embodiment, the therapeutic agent is cyclosporine A.
In one embodiment, the therapeutic agent is dorzolamide. In one
embodiment, the therapeutic agent is natamycin. In one embodiment,
the therapeutic agent is olopatadine.
[0169] In some embodiments, the therapeutic agent is an antibiotic,
for example, a fluoroquinolone, vancomycin, cephalosporin,
gentamycin, erythromycin, azithromycin, a sulfa drug, bacitracin,
gatifloxacin, levofloxin, moxifloxacin, or ofoxacin.
[0170] In some embodiments, the therapeutic agent is an antiviral,
for example, acyclovir, gancyclovir, valcyclovir.
[0171] In some embodiments, the therapeutic agent is an antiallergy
agent, for example, an antihistamine. In one embodiment, the
therapeutic agent is olopatadine.
[0172] In some embodiments, the nanoparticle composition comprising
the therapeutic agent is administered to the anterior surface of
the eye.
[0173] In some embodiments, the ophthalmic agent is formulated in a
dosage form for administration to the eye surface, such as a drop,
ointment or gel. In some embodiments, the ophthalmic agent is
formulated in a dosage form for administration to the eye via a
contact lens.
[0174] Diagnostic Agent
[0175] In another embodiment, the payload is a diagnostic agent.
For example, the payload may be a fluorescent molecule; a gas; a
metal; a commercially available imaging agent used in positron
emissions tomography (PET), computer assisted tomography (CAT),
single photon emission computerized tomography, x-ray, fluoroscopy,
and magnetic resonance imaging (MRI); or a contrast agents.
Non-limiting examples of suitable materials for use as contrast
agents in MRI include gadolinium chelates, as well as iron,
magnesium, manganese, copper, and chromium. Examples of materials
useful for CAT and x-ray imaging include, but are not limited to,
iodine-based materials.
[0176] Radionucleotide
[0177] As another example, the payload may include a radionuclide,
e.g., for use as a therapeutic, diagnostic, or prognostic agent.
Among the radionuclides used, gamma-emitters, positron-emitters,
and X-ray emitters are suitable for diagnostic and/or therapy,
while beta emitters and alpha-emitters may also be used for
therapy. Suitable radionuclides for use with various embodiments of
the present invention include, but are not limited to, .sup.123I,
.sup.125I, .sup.130I, .sup.131I, .sup.133I, .sup.135I, .sup.47Sc,
.sup.72As, .sup.72Sc, .sup.90Y, .sup.88Y, .sup.97Ru, .sup.100Pd,
.sup.101mRh, .sup.119Sb, .sup.128Ba, .sup.197Hg, .sup.211At,
.sup.212Bi, .sup.212Pb, .sup.109Pd, .sup.111In, .sup.67Ga,
.sup.68Ga, .sup.67Cu, .sup.75Br, .sup.77Br, .sup.99mTc, .sup.14C,
.sup.13N, .sup.15O, .sup.32P, .sup.33P, or .sup.18F. The
radionucleotide may be contained within the nanoparticle (e.g., as
a separate species), and/or form part of a macromolecule or polymer
that forms the nanoparticle.
[0178] Pharmaceutical Compositions
[0179] Another aspect of the disclosure is related to
pharmaceutical compositions comprising a nanoparticle composition
as defined herein, and a pharmaceutically acceptable carrier.
Pharmaceutical compositions can be prepared in a manner well known
in the pharmaceutical art, and can be administered by a variety of
routes of administration, depending upon whether local or systemic
effect is desired and upon the area to be treated.
[0180] In some embodiments, the pharmaceutical composition is
administered to a desired mucosal site in a subject. The
pharmaceutical composition can be administered to a desired mucosal
site by any suitable route of administration. In some embodiments,
the route of administration is non-parenteral, such as topical. As
used herein, topical administration may include, for example,
administration to a mucous membrane via the mouth, eye, ear, nose,
esophagus, stomach, small intestine, large intestine, rectum,
vagina, urethra, penis, uterus, etc. It is understood that
administration of a therapeutic agent to a mucosal site may provide
local and/or systemic effect, for example, depending on the ability
of the agent to be absorbed into the circulation via the mucous
membrane.
[0181] Pharmaceutical compositions and formulations for topical
administration generally include ointments, lotions, creams, gels,
drops, suppositories, sprays, liquids and powders. For topical
administration to a mucous membrane of the gut, an oral dosage form
such as a liquid, emusion, tablet, caplet or capsule may be used.
Conventional pharmaceutical carriers, excipients and dulients may
be employed.
[0182] In some embodiments, the compositions are administered in a
dosage form suitable for topical or transdermal administration.
Non-limiting examples of dosage forms suitable for topical or
transdermal administration of a pharmaceutical composition as
disclosed herein include ointments, pastes, creams, lotions, gels,
powders, solutions, suspensions, emulsions, sprays, inhalants, or
patches. The composition is typically admixed under sterile
conditions with a pharmaceutically acceptable carrier and any
needed preservatives or buffers as may be required.
[0183] In some embodiments, the composition is in a dosage form
suitable for oral administration. Such a dosage form may, for
example, be useful for administration to an oral, esophageal,
gastric or intestinal mucosal site. The composition may or may not
be swallowed depending on the target mucosal site. For example, the
dosage form could be a mouth wash. In some embodiments, the oral
dosage form is a liquid dosage form, such as a suspension, solution
or emulsion. In some embodiments, the dosage form is a solid dosage
from, such as a powder, tablet, capsule or caplet.
[0184] In some embodiments, the composition is in a dosage form
suitable for rectal or vaginal administration. In some embodiments,
the composition for rectal or vaginal administration is in the form
of a suppository. In some embodiments, the composition for rectal
or vaginal administration is in the form of a liquid, such as a
douche or enema. In some embodiments, the composition for rectal or
vaginal administration is in the form of a cream, ointment or gel,
which may optionally be applied using an applicator.
[0185] In some embodiments, the composition is in a dosage form
suitable for nasal or pulmonary administration. In some
embodiments, the dosage form for nasal or pulmonary administration
is a spray or inhalant. In one embodiment, the dosage form is a
spray. In one embodiment, the dosage form in an inhalant, which may
be administered with an inhaler.
[0186] In some embodiments, the composition is in a dosage form
suitable for ocular or otic administration, i.e. administration to
the eye or ear. In some embodiments, the dosage form for ocular or
otic administration is a drop. In some embodiments, the composition
is in a dosage form suitable for ocular administration, such as a
drop, gel or ointment. Such drop, gel or ointment may, for example,
be applied to the anterior surface of the eye.
[0187] Parenteral routes of administration includes intravenous,
intraarterial, subcutaneous, intraperitoneal intramuscular or
injection or infusion; or intracranial, e.g., intrathecal or
intraventricular, administration. Parenteral administration can be
in the form of a single bolus dose, or may be, for example, by a
continuous perfusion pump.
[0188] In some embodiments, parenteral routes are desirable since
they avoid contact with the digestive enzymes that are found in the
alimentary canal. According to such embodiments, the nanoparticle
compositions may be administered by injection (e.g., intravenous,
subcutaneous or intramuscular, intraperitoneal injection),
rectally, vaginally, topically (as by powders, creams, ointments,
or drops), or by inhalation (as by sprays).
[0189] Injectable preparations, for example, sterile injectable
aqueous or oleaginous suspensions may be formulated according to
the known art using suitable dispersing or wetting agents and
suspending agents. The sterile injectable preparation may also be a
sterile injectable solution, suspension, or emulsion in a nontoxic
parenterally acceptable diluent or solvent, for example, as a
solution in 1,3-butanediol. Among the acceptable vehicles and
solvents that may be employed are water, Ringer's solution, U.S.P.,
and isotonic sodium chloride solution. In addition, sterile, fixed
oils are conventionally employed as a solvent or suspending medium.
For this purpose any bland fixed oil can be employed including
synthetic mono- or diglycerides. In addition, fatty acids such as
oleic acid are used in the preparation of injectables. In one
embodiment, the inventive conjugate is suspended in a carrier fluid
comprising 1% (w/v) sodium carboxymethyl cellulose and 0.1% (v/v)
TWEEN.TM. 80. The injectable formulations can be sterilized, for
example, by filtration through a bacteria-retaining filter, or by
incorporating sterilizing agents in the form of sterile solid
compositions which can be dissolved or dispersed in sterile water
or other sterile injectable medium prior to use.
[0190] Ointments, pastes, creams, and gels may contain, in addition
to the nanoparticle delivery system of the present disclosure,
excipients such as animal and vegetable fats, oils, waxes,
paraffins, starch, tragacanth, cellulose derivatives, polyethylene
glycols, silicones, bentonites, silicic acid, talc, and zinc oxide,
or mixtures thereof.
[0191] Powders and sprays can contain, in addition to the inventive
conjugates of this invention, excipients such as lactose, talc,
silicic acid, aluminum hydroxide, calcium silicates, and polyamide
powder, or mixtures thereof. Sprays can additionally contain
customary propellants such as chlorofluorohydrocarbons.
[0192] Pharmaceutical compositions for oral administration can be
liquid or solid. Liquid dosage forms suitable for oral
administration of inventive compositions include pharmaceutically
acceptable emulsions, microemulsions, solutions, suspensions,
syrups, and elixirs. In addition to an encapsulated or
unencapsulated conjugate, the liquid dosage forms may contain inert
diluents commonly used in the art such as, for example, water or
other solvents, solubilizing agents and emulsifiers such as ethyl
alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl
alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol,
dimethylformamide, oils (in particular, cottonseed, groundnut,
corn, germ, olive, castor, and sesame oils), glycerol,
tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid
esters of sorbitan, and mixtures thereof. Besides inert diluents,
the oral compositions can also include adjuvants, wetting agents,
emulsifying and suspending agents, sweetening, flavoring, and
perfuming agents. As used herein, the term "adjuvant" refers to any
compound which is a nonspecific modulator of the immune response.
In certain embodiments, the adjuvant stimulates the immune
response. Any adjuvant may be used in accordance with the present
invention. A large number of adjuvant compounds is known in the art
(Allison Dev. Biol. Stand. 92:3-11, 1998; Unkeless et al. Annu.
Rev. Immunol. 6:251-281, 1998; and Phillips et al. Vaccine
10:151-158, 1992).
[0193] Solid dosage forms for oral administration include capsules,
tablets, caplets, pills, powders, and granules. In such solid
dosage forms, the encapsulated or unencapsulated conjugate is mixed
with at least one inert, pharmaceutically acceptable excipient or
carrier such as sodium citrate or dicalcium phosphate and/or (a)
fillers or extenders such as starches, lactose, sucrose, glucose,
mannitol, and silicic acid, (b) binders such as, for example,
carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone,
sucrose, and acacia, (c) humectants such as glycerol, (d)
disintegrating agents such as agar-agar, calcium carbonate, potato
or tapioca starch, alginic acid, certain silicates, and sodium
carbonate, (e) solution retarding agents such as paraffin, (f)
absorption accelerators such as quaternary ammonium compounds, (g)
wetting agents such as, for example, cetyl alcohol and glycerol
monostearate, (h) absorbents such as kaolin and bentonite clay, and
(i) lubricants such as talc, calcium stearate, magnesium stearate,
solid polyethylene glycols, sodium lauryl sulfate, and mixtures
thereof. In the case of capsules, tablets, and pills, the dosage
form may also comprise buffering agents.
[0194] Solid compositions of a similar type may also be employed as
fillers in soft and hard-filled gelatin capsules using such
excipients as lactose or milk sugar as well as high molecular
weight polyethylene glycols and the like. The solid dosage forms of
tablets, dragees, capsules, pills, and granules can be prepared
with coatings and shells such as enteric coatings and other
coatings well known in the pharmaceutical formulating art.
[0195] Dosage
[0196] It will be appreciated that the exact dosage of the
nanoparticle or components thereof, such as a therapeutic agent,
may be determined by a physician in view of the patient to be
treated. In general, dosage and administration are adjusted to
provide an effective amount of the inventive conjugate to the
patient being treated. As used herein, the "effective amount"
refers to the amount necessary to elicit the desired biological
response. As will be appreciated by those of ordinary skill in this
art, the effective amount may vary depending on such factors as the
desired biological endpoint, the drug to be delivered, the target
tissue, the route of administration, etc. Additional factors which
may be taken into account include the severity of the disease
state; age, weight and gender of the patient being treated; diet,
time and frequency of administration; drug combinations; reaction
sensitivities; and tolerance/response to therapy.
[0197] The compositions described herein may be formulated in unit
dosage form for ease of administration and uniformity of dosage.
The expression "unit dosage form" as used herein refers to a
physically discrete unit appropriate for the patient to be treated.
It will be understood, however, that the total daily dosage of the
composition of the present invention may be decided by a physician.
For any composition, the therapeutically effective dose can be
estimated initially either in cell culture assays or in animal
models, usually mice, rabbits, dogs, or pigs. The animal model is
also used to achieve a desirable concentration range and route of
administration. Such information can then be used to determine
useful doses and routes for administration in humans. Therapeutic
efficacy and toxicity of conjugates can be determined by standard
pharmaceutical procedures in cell cultures or experimental animals,
e.g., ED50 (the dose is therapeutically effective in 50% of the
population) and LD50 (the dose is lethal to 50% of the population).
The dose ratio of toxic to therapeutic effects is the therapeutic
index, and it can be expressed as the ratio, LD50/ED50.
Pharmaceutical compositions which exhibit large therapeutic indices
may be useful in some embodiments. The data obtained from cell
culture assays and animal studies can be used in formulating a
range of dosage for human use.
[0198] Kits and Commercial Packages
[0199] The present disclosure also provides any of the
above-mentioned compositions in kits or commercial packages,
optionally with instructions for use or administration of any of
the compositions described herein by any suitable technique as
previously described. "Instructions" can define a component of
promotion, and typically involve written instructions on or
associated with packaging of compositions of the invention.
Instructions also can include any oral or electronic instructions
provided in any manner. The "kit" typically defines a package
including any one or a combination of the compositions of the
invention and the instructions, but can also include the
composition of the invention and instructions of any form that are
provided in connection with the composition in a manner such that a
clinical professional will clearly recognize that the instructions
are to be associated with the specific composition.
[0200] The kits described herein may also contain one or more
containers, which may contain the inventive composition and other
ingredients as previously described. The kits also may contain
instructions for mixing, diluting, and/or administrating the
compositions of the invention in some cases. The kits also can
include other containers with one or more solvents, surfactants,
preservative and/or diluents (e.g., normal saline (0.9% NaCl), or
5% dextrose) as well as containers for mixing, diluting or
administering the components in a sample or to a subject in need of
such treatment.
[0201] The compositions of the kit may be provided as any suitable
form, for example, as liquid solutions or as dried powders. When
the composition provided is a dry powder, the composition may be
reconstituted by the addition of a suitable diluent, which may also
be provided. In embodiments where liquid forms of the composition
are used, the liquid form may be concentrated or ready to use. The
diluent will depend on the components of the composition and the
mode of use or administration. Suitable diluents for drug
compositions are well known, for example as previously described,
and are available in the literature. The diluent will depend on the
conjugate and the mode of use or administration.
[0202] The present disclosure also encompasses, in another aspect,
promotion of the administration of the nanoparticle delivery system
described herein. In some embodiments, one or more compositions of
the invention are promoted for the prevention or treatment of
various diseases such as those described herein via administration
of any one of the compositions of the present invention. As used
herein, "promoted" includes all methods of doing business including
methods of education, hospital and other clinical instruction,
pharmaceutical industry activity including pharmaceutical sales,
and any advertising or other promotional activity including
written, oral and electronic communication of any form, associated
with compositions of the invention.
[0203] Methods of Treatment and Use
[0204] The nanoparticle compositions disclosed herein may be useful
in the treatment or prevention of any disease or condition capable
of being treated via controlled delivery of a therapeutic agent to
a mucosal site. As used herein "treating" includes preventing,
reducing or alleviating one or more signs and/or symptoms of the
disease or condition. The nanoparticle compositions provide
controlled release of the therapeutic agent and are
surface-functionalized for targeting and retention of the
nanoparticles at the mucosal site such that sustained release at
the mucosal site can be achieved.
[0205] In some embodiments, there are provided methods of treating
a disease or condition in a subject by administering an effective
amount of a composition or component thereof as defined herein. In
other embodiments, there are provided uses of the compositions or
components thereof as defined herein for treating and/or preventing
a disease or condition. In other embodiments, there are provided
uses of the compositions or components thereof as defined herein
for the manufacture of a medicament to treating and/or preventing a
disease or condition. In other embodiments, there are provided
compositions or components thereof disclosed herein for the
manufacture of a medicament to treating and/or preventing a disease
or condition. In other embodiments, there are provided compositions
or components thereof as defined herein for the treatment of a
disease or condition.
[0206] In some embodiments, the disease or condition to be treated
is a disease or condition capable of being treated via delivery of
a therapeutic agent to a mucosal site, such as a mucosal site of
the mouth, eye, ear, nose, esophagus, stomach, small intestine,
large intestine, rectum, vagina, urethra, penis or uterus. In some
embodiments, the disease or condition to be treated is a disease or
condition affecting the mouth, eye, ear, nose, esophagus, stomach,
small intestine, large intestine, rectum, vagina, urethra, penis,
or uterus. In some embodiments, the disease or condition to be
treated is a disease or condition affecting the mouth, eye, ear or
nose. In some embodiments, the disease or condition to be treated
is a disease or condition affecting the rectum, vagina, urethra,
penis, or uterus. In some embodiments, the disease or condition to
be treated is a disease or condition affecting the esophagus,
stomach, small intestine or large intestine.
[0207] In some embodiments, the disease or condition to be treated
is a disease or condition affecting the eye. Non-limiting examples
include abrasion, acanthamoeba keratitis, actinic keratosis, acute
allergic blepharoconjunctivitis, allergic conjunctivitis,
adenoviral keratoconjunctivitis, aniridia, atopic
keratoconjunctivitis, bacterial conjunctivitis, bacterial
keratitis, band keratopathy, basal cell carcinoma, Bell's palsy,
blepharitis, bullous keratopathy, canaliculitis, caruncular cyst,
cataract, chalazion, chlamydial conjunctivitis, climatic droplet
keratopathy, concretions, conjunctival intraepithelial neoplasia,
conjunctival lymphoma, conjunctival papilloma, conjunctival
pigmented lesions, conjunctival scarring, conjunctivitis,
conjunctivochalasia and chemosis, corneal collagen cross-linking,
corneal edema, corneal graft--lamellar keratoplasty, corneal graft
rejection, corneal infiltrates, crocodile shagreen, crystalline
keratopathy, cysts of the eye lids, dacryocystitis, dellen,
dendritic ulcer, dermatochalasis and blepharochalasis, Descernet's
membrane breaks, disciform keratitis, disciform keratitis,
keratoconjunctivitis sicca, ectopia lentis, ectropion,
endophthalmitis, entropion, epiblepharon and epicanthic folds,
epibulbar choristomas, epiphora, episcleritis, epithelial and
fibrous ingrowth, epithelial basement membrane dystrophy, exposure
keratopathy, eyelid trauma, filamentary keratopathy, filtering
bleb, flash burns, floppy eyelid syndrome, follicular
conjunctivitis, Fuchs' endothelial dystrophy, Fuchs' heterochromic
iridocyclitis, fungal keratitis, giant papillary conjunctivitis,
glaucoma--acute angle closure, gonococcal keratoconjunctivitis,
granular dystrophy, hemangioma, herpes simplex keratitis, herpes
simplex primary blepharokeratoconjunctivitis, herpes zoster
ophthalmicus, hordeolum--internal and external, hyphema--blunt
trauma, hypopyon, infectious crystalline keratopathy, interstitial
keratitis, iridocorneal dysgenesis, iridocorneal endotheliopathy,
iris cysts, iritis, iron lines, keratoconus, keratoconus forme
frusta, keratoglobus, lattice stromal dystrophy, leukocoria, lice,
limbal stem cell deficiency, lipid keratopathy, macular stromal
dystrophy, marginal keratitis, meesmann's dystrophy,
melanoma--conjunctival and eyelid, melanoma and nevus of the iris,
membranous and pseudomembranous conjunctivitis, molluscum
contagiosum, mooren's ulcer, nasolacrimal duct
obstruction--congenital, neurotrophic keratopathy, nevus--eyelid,
ocular cicatricial pemphigold, ophthalmia neonatorum, pannus and
pseudopterygia, pellucid marginal degeneration,
perforation--corneal, peripheral ulcerative keratitis, persistent
epithelial defect, phlyctenulosis, pingueculum, posterior capsular
opacification, posterior polymorphous dystrophy, preseptal
cellulitis, pseudoexfoliation of the lens capsule, pterygium,
ptosis and pseudoptosis, punctual stenosis, pyogenic granuloma,
recurrent corneal erosion syndrome, Reis-Buckler's dystrophy,
retention cyst and lymphangiectasia, rheumatoid arthritis, rosacea
keratitis, Salzmann nodular degeneration, scleritis, sebaceous cell
carcinoma, seborrheic keratosis, squamous cell carcinoma--lid,
Stevens-Johnson syndrome, sub-conjunctival hemorrhage, superficial
punctate keratopathy, superior limbic keratoconjunctivitis,
synechia, Terrien's marginal degeneration, Thygeson's superficial
punctate keratopathy, toxic keratopathy, trachoma, trichiasis,
pseudotrichiasis, distachiasis, metaplastic lashes,
trichotillomania, uveitis, vernal keratoconjunctivitis, vitamin A
deficiency, vortex keratopathy, xanthelasma.
[0208] In some embodiments, the disease or condition is a disease
or condition of the eye is glaucoma, keratoconjunctivitis sicca or
allergic conjunctivitis, fungal infection, viral infection or
bacterial infection. In some embodiments, the disease or condition
of the eye is glaucoma, keratoconjunctivitis sicca or allergic
conjunctivitis. In some embodiments, the disease or condition of
the eye is a fungal infection, viral infection or bacterial
infection.
[0209] In some embodiments, Cyclosporine A is administered for the
treatment of keratoconjunctivitis sicca. In some embodiment,
Olopatadine is administered for the treatment of allergic
conjunctivitis. In some embodiment, Brinzolamide, Brimonidine, or
Dorzolamide, are administered for the treatment of glaucoma.
[0210] In some embodiments, the composition is administered
topically on the surface of the eye for treatment of diseases
associated with the anterior segments of the eye.
[0211] In some embodiments, the composition is administered
topically on the surface of the eye for treatment of diseases
associated with the posterior segments of the eye. In some
embodiments, the composition is administered intranasally to target
the nasal mucosa. In some embodiments, the composition is
administered orally to target the oral mucosa. In some embodiments,
the composition is administered intravenously to target the
gastrointestinal mucin for treatment of diseases associated with
the intestine. In some embodiments, the composition is administered
vaginally to target the vaginal mucosa. In some embodiments, the
composition administered rectally to target the rectal mucosa.
[0212] In some embodiments, the disease or condition to be treated
is selected from one or more of acquired angioedema, acrodermatitis
enteropathica, acute serous conjunctivitis, adenomatous polyposis
of the colon, adenoviridae infections, adenovirus-related cold,
allergic asthma, allergic contact cheilitis, allergic rhinitis,
allergies, amyloidosis of gingiva and conjunctiva mental
retardation, analgesic asthma syndrome, Anderson's triad, angina
bullosa haemorrhagica, angular conjunctivitis, asthma, asthmatic
Bronchitis, atrophic glossitis, atrophic rhinitis, attenuated
familial polyposis, Behcet's disease, benign migratory glossitis,
benign mucosal penphigoid, black hairy tongue, Brodie pile,
bronchitis, bullous penphigoid, candidiasis, canker sores, carbon
baby syndrome, cariomegaly, catarrh, catarrhal or mucopurulent
conjunctivitis, central papillary atrophy, cervical polyps,
cheilitis, cheilitis exfoliativa, cheilitis glandularis, cheilitis
granulomatosa, cholecystitis, cicatrizing conjunctivitis, ciliary
discoordination due to random ciliary orientation, ciliary
dyskinesia, colitis, colorectal adenomatous polyposis, colorectal
polyps, conjunctivitis ligneous, conjunctivitis with
pseudomembrane, coronavirus-related cold, costello syndrome,
coxsackievirus-related cold, Crohn's disease, cronkhite-Canada
syndrome, cystic Fibrosis, cystitis, dermatostomatitis,
desquamative gingivitis, dextrocardia-bronchiectasis-sinusitis,
drug-induced ulcer of the lip, duodenal ulcer, dyskeratosis
congenital, dyskeratosis congenita of Zinsser-Cole-Engman,
echovirus-related cold, Ectodermal dysplasia, enterocolitis,
eosinophilic cystitis, epidemic kaposi's sarcoma, epulis, epulis
fissuratum, eruptive hemangioma, eruptive lingual papillitis,
erythroplakia, esophageal ulcer, esophagitis, extrinsic asthma,
familial adenomatous polyposis, familial intestinal polyposis,
familial nasal acilia, familial polyposis, Fenwick ulcer, fissured
tongue, flu, folicular conjunctivitis, follicular hamartoma, food
allergy related asthma, Fordyce's disease, Gardner syndrome,
gastresophageal reflux-related chronic cough, gastric erosion,
gastric reflux, gastric ulcer, gastritis, gastritis,
gastroesophageal reflux disease, giant papillary conjunctivitis,
gonorrhea, growth-hormone secreting pituitary adenoma, hairy
leukoplakia, hemophilus influenzae B, hemorrhagic conjunctivitis,
hemorrhagic proctocolitis, herpes, human papillomavirus, immotile
cilia syndrome, inclusion conjunctivitis, influenza A, influenza B,
interstitial cystitis, intraoral dental sinus, intrinsic asthma,
invasive candidiasis, irritative conjunctivitis,
Jadassohn-Lewandowsky syndrome, kaposiform hemangio-endothelioma,
keratoconjunctivitis, keratosis pharynges, laryngopharyngeal
reflux, leprosy, leukoencephalopathy, leukoplakia, leukoplakia with
tylosis and esophageal carcinoma, lipogranulomatosis, logic
syndrome, lower esophageal ulcer, lymphocytic colitis, lymphoma,
mucosa-associated lymphoid tissue, major ulcerative stomatitis,
malignant peptic ulcer, Melkersson-Rosenthal syndrome, membranous
conjunctivitis, mouth ulcers, mucinous carcinoma, mucocele,
mucoepidermoid, mucoepidermoid carcinoma, mucoepithelial dysplasia,
Witkop type, mucosal leishmaniasis, mucosal lichen planus, mucosal
squamous cell carcinoma, mucositis, mucous cyst of oral mucosa,
Nagayama's spots, nasal polyp, necrotizing entercolitis,
necrotizing periodontal diseases, nicotine stomatitis, ophthalmia
neonatorum, oral Crohn's disease, oral florid papillomatosis, oral
fordyce granules, oral thrush, oral ulcer, orthomyxovirus-related
cold, Osler-Rendu-Weber syndrome, pancolitis, papillary
conjunctivitis, parainfluenza, paramyxovirus-related cold,
paucigranulocytic asthma, pemphigus, pemphigus foliaceus, pemphigus
volgaris, Penign peptic ulcer, penphigus vulgaris, peptic ulcer,
periadenitis mucosa necrotica, periodic fever, pharyngoconjunctival
fever, Pinguecula, plasma cell cheilitis, plasmoacanthoma/plasma
cell gingivitis, primary ciliary dyskinesia, proctitis
pseudomembranous colitis, pseudomycoma peritonei, psoriasis on
mucous membranes, psychiatric disorders associated celiac disease,
pterygium, pterygium of the conjunctiva, purulent conjunctivitis,
recurring scarring aphthae, reflux laryngitis, refractory celiac
disease, Rhinitis, rhinosporidiosis, ritter syndrome, rostan
asthma, salicylate-sensitive asthma, Schafer syndrome, sinusitis,
Sjogren syndrome, spring catarrh, sprue, Stevens-Johnson syndrome,
stomal ulcer, stomatitis, superior limbic keratoconjunctivitis,
Sutton disease, swime flu, systemic candidiasis, Takahara's
disease, the clap, thrush, trumpeter's wart, tuberculous disease of
the mucous, ulcerative colitis, ulcerative conjunctivitis,
ulcerative proctosigmoiditis, urban Schosser Spohn synfrome,
vaginal candidiasis, vasomotor rhinitis, vestibular papillomatosis,
Vincent's angina, vulvovaginal gingival syndrome, white sponge
nevus, xanthogranulomatous cholecystitis, xerostomia
[0213] Subject
[0214] The subject may be a human or non-human animal. In some
embodiments, the subject is a mammal. Non-limiting examples of
mammals include human, dog, cat, horse, donkey, rabbit, cow, pig,
sheep, goat, rat, mouse, guinea pig, hamster, and primate. In some
embodiments, the subject is a human.
[0215] Methods of Manufacture
[0216] In another aspect, the present disclosure provides a process
for the preparation of macromolecules useful in the formation of a
mucoadhesive nanoparticle delivery system. The macromolecule is
typically an amphiphilic copolymer, in particular, a block
copolymer, which is conjugated to a plurality of mucosal targeting
moieties. The macromolecules are capable of assembly under suitable
conditions to form a nanoparticle, i.e. of the core-shell type. In
an aqueous environment, the nanoparticle has a hydrophobic core and
a hydrophilic shell, the shell providing a surface of the
nanoparticle, the surface of the nanoparticle being coated in a
desired amount (i.e. surface density) of the mucosal targeting
moiety for controlled targeting and adhesion of the
nanoparticle.
[0217] The macromolecules disclosed herein may be made by any
suitable process known to those skilled in the art, for example,
using suitable conjugation techniques. Starting materials,
including hydrophobic polymer and hydrophilic polymer, may be
purchased from various commercial suppliers. Where desired, the
starting materials can be prepared by those of skill in the art.
For example, where polymers comprising modified backbone residues
are desired. Exemplary methods for making macromolecules useful in
the formation of a mucoadhesive delivery system are described
below.
[0218] In some embodiments, there is provided a method of preparing
a nanoparticle composition.
[0219] In some embodiments, the method is carried out in a series
of steps, such as, preparation of an amphiphilic macromolecule,
nanoparticle formation, and conjugation to a targeting moiety (i.e.
coating of the surface of the nanoparticle with a desired surface
density of the targeting moiety). Alternatively, the hydrophilic
portion comprising multiple functional groups may first be coupled
to a desired amount of the targeting moiety, followed by
conjugation of the functionalized hydrophilic portion to a
hydrophobic polymer, which may be in the form of a hydrophobic
nanoparticle (i.e. coating the surface of a hydrophobic
nanoparticle with a functionalized hydrophilic polymer). When the
hydrophobic polymer is modified for conjugation, one end of the
polymer will typically become more hydrophilic (e.g. presence of a
carboxyl group). Such polymers can assemble to form hydrophobic
nanoparticles in aqueous medium. Preparation of the nanoparticles
in a controlled sequence results in surface-functionalized
nanoparticles wherein substantially all (e.g. greater than 90%,
95%, 96%, 97%, 98%, 99%) of the targeting moieties are located on
the surface of the nanoparticle formed by the hydrophilic portion
of the macromolecules.
[0220] In one embodiment, the method of preparing a nanoparticle
composition useful for delivery of a payload to a mucosal site
comprises preparing an ampliphilic macromolecule comprising a
hydrophilic portion and a hydrophobic portion, the hydrophilic
portion comprising multiple functional moieties; b) assembling a
plurality of said macromolecules under suitable conditions to form
a nanoparticle having a hydrophobic core and a hydrophilic shell;
and c) conjugating at least a portion of said functional moieties
on the hydrophobic portion to a mucosal targeting moiety, to
thereby provide a surface-functionalized nanoparticle.
[0221] In some embodiments, a) comprises conjugation of a
hydrophilic polymer to a hydrophobic polymer to form a diblock
copolymer.
[0222] In some embodiments, the hydrophilic polymer is dextran and
the hydrophobic polymer is PLA.
[0223] In some embodiments, the targeting moiety is a phenylboronic
acid derivative, a thiol derivative or an acrylate derivative. In
some embodiments, the targeting moiety is a phenylboronic acid
(PBA) derivative.
[0224] In some embodiments, step b) is performed before step c).
However, in other embodiments, step c) is performed before step
b).
[0225] In some embodiments, the surface density of the mucosal
targeting moiety on the nanoparticle is controlled by the amount of
mucosal targeting moiety introduced into the reaction.
[0226] In some embodiments, the process comprises reductive
animation between the multimer and a suitable linker. In some
embodiments, the reaction takes place between the amine end of
N-protected-ethylenediamine and the reducing end of a multimer
having multiple functional groups per monomer unit, such as a
polysaccharide, a polynucleotide or a polypeptide. Any suitable
N-protecting group can be used. In some embodiments, the
N-protecting group is tert-butoxycarbonyl (BOC).
[0227] The choice of a hydrophilic polymer having multiple
functional groups per monomer unit enables tuning of the resulting
nanoparticle to control particle size, targeting and/or adhesion at
a mucosal site, as described further below. In some embodiments,
the hydrophilic polymer is a polysaccharide. In some embodiments,
the hydrophilic polymer is dextran. Therefore, in some embodiments,
the reaction takes place between N--BOC-ethylenediamine and an
aldehyde of the reducing end of a dextran polymer. The reaction may
be carried out in a suitable solvent, such as a borate buffer
solution, in the presence of a reducing agent, such as
NaCNBH.sub.3. The mixture is stirred for a sufficient amount of
time to complete the reaction, for example, about 24 to 120 hours
hours. In one embodiment the mixture is stirred for about 24, 48,
72, 96, or 120 hours. In some embodiments, this step is carried out
at room temperature. In some embodiments, this step is carried out
in the dark.
[0228] The mixture may then be washed to remove any unreacted
molecules or catalysts. In one embodiment methanol is used in the
washing step. The end-modified dextran can optionally be dried
before continuing the process.
[0229] The protecting group is then removed followed by conjugation
of the amine-terminated multimer to a hydrophobic polymer in a
suitable solvent to provide an amphiphilic macromolecule. In one
embodiment, hydrochloric acid and triethyl amien are used for the
removal of the protecting group. The macromolecule may be washed,
for example, using methanol, to remove unreacted polymer.
[0230] The conjugation of the amine-terminated multimer with a
hydrophobic polymer takes place in a suitable solvent. In one
embodiment, the solvent is DMSO, acetone, or acetonitrile.
Catalysts may be employed to drive the reaction. In one embodiment,
the catalysts are EDC and Sulfo-NHS.
[0231] The mixture may then be washed to remove any unreacted
molecules or catalysts. In one embodiment methanol is used in the
washing step. Additional washing step may be used to remove
unreacted polymer. In one embodiment, the unreacted polymer is
dextran. The final mixture is dissolved in a suitable solvent,
centrifuged and the resulting supernatant is collected. In one
embodiment, the suitable solvent is acetone or acetonitrile. The
final product is dried. In one embodiment, vacuum dessicator is
used to dry the product.
[0232] In another aspect, the present disclosure provides a process
for the preparation of nanoparticles useful in the formation of a
mucoadhesive nanoparticle delivery system. The polymers or
macromolecules described herein may be formed into a nanoparticle
using techniques known to those skilled in the art. The geometry
formed by the particle from the macromolecule may depend on factors
such as the size and composition of the polymers that form the
macromolecule. In addition, also as discussed below, in some cases,
the particle may include an agent of interest, such as a
therapeutic, diagnostic or imaging agent. For example, in some
embodiments, the nanoparticle may contain a therapeutic agent, such
as a drug. The agent of interest may be incorporated into the
particle during formation of the particle, e.g., by including the
agent in a solution containing the polymers that are used to form
the particle, and/or the agent may be incorporated in the particle
after its formation.
[0233] In addition, the method may employ additional polymers or
macromolecules distinguishable from the polymers or macromolecules
discussed above. As previously discussed, first and second (or
more) macromolecules may be combined together at different ratios
to produce particles comprising the first and second (or more)
macromolecules, keeping in mind that, in some embodiments, it is
desirable to have hydrophilic portions with multiple functional
groups present in the shell of the nanoparticle for tunable
targeting of the nanoparticles via coupling of the functional
groups to a mucosal targeting moiety, such as a targeting moiety
capable of forming high affinity binding to a target at the mucosal
site.
[0234] In some embodiments, the targeting moieties are conjugated
to the macromolecules following nanoparticle formation.
[0235] The present disclosure also provides a process for
conjugating targeting moieties on the surface of the nanoparticles
formed using the amphiphilic macromolecules described herein. In
some embodiments, the conjugation is between the functional groups
of the hydrophilic portion (e.g. the backbone of a hydrophilic
polymer) and the functional groups of the targeting moieties. In
some embodiment, catalysts, for example, EDC, are used for the
conjugation reaction. In some embodiments, the functional groups of
the polymer backbone are modified into other types of functional
groups prior to the conjugation reaction. In one embodiment,
NaIO.sub.14 is used to oxidize hydroxyl groups into aldehyde
groups. The mixture may be washed, for example, using methanol, to
remove nonconjugated targeting moieties. In some embodiment,
dialysis is used to remove the unreacted molecules.
[0236] In some cases, the method may include conjugation with more
than one type of targeting moiety. The surface density of targeting
moiety on the resulting nanoparticles may be controlled by
adjusting the amount of material in the reaction mixture.
[0237] In some embodiments, conjugation and nanoparticle formation
may occur as a single-step reaction, for example, according to a
single-step reaction as described in U.S. Pat. No. 8,323,698.
However, a single-step reaction such as this will result in a
nanoparticle having a detectable amount of targeting moieties
located within the core of the nanoparticle, thereby decreasing the
targeting efficiency of the particles compared to a more controlled
sequence as described above.
[0238] Specific reaction conditions can be determined by those of
ordinary skill in the art using no more than routine
experimentation.
[0239] Embodiment of the Method
[0240] An exemplary method of preparing a Dextran-b-PLA (Dex-b-PLA)
block copolymer is described below in Example 1 (Verma, 2012).
Briefly, an exemplary procedure for the synthesis of Dex-b-PLA may
be divided into three stages: reductive amination between Dextran
and N-Boc-ethylenediamine, deprotection of the Boc group, and
conjugation of the amine-modified Dextran end group with
carboxyl-terminated PLA. Reductive amination may be carried out by
dissolving Dex in a borate buffer and mixing it with
N-Boc-ethylenediamine and NaCNBH.sub.3 in dark condition for about
72 hrs. After the reaction, the mixture is washed with methanol and
dried in vacuum desiccator. The sample is then dissolved in
DI-H.sub.2O and treated with hydrochloric acid and triethyl amine
for the deprotection of the Boc group. The conjugation of
amine-terminated Dextran and PLA was carried out in DMSO with EDC
and Sulfo-NHS as catalysts for about 4 hrs. The final product was
washed several times with methanol. The wash sample was further
dissolved in acetone and centrifuged. The supernatant was extracted
carefully in order to separate from free unreacted Dextran that
have been precipitated. Finally, the supernatant containing
Dex-b-PLA was dried in vacuum desiccator.
[0241] Thus, in some embodiments, there is provided a method of
preparing a Dex-b-PLA macromolecule, the method comprising: 1)
reductive amination between Dextran and N-Boc-ethylenediamine, 2)
deprotection of the Boc group, and 3) conjugation of the end
modified Dextran with PLA (Scheme 1). The first step of the
synthesis involves reductive amination between the aldehyde on the
reducing end of Dextran and the amine group of
N-Boc-ethylenediamine cross-linker. The reducing agent,
NaCNBH.sub.3 was added to the borate buffer solution and the
mixture was stirred for 72 hours in dark conditions at room
temperature. The mixture was then washed in methanol to remove any
unreacted molecules or catalysts. The end-modified Dextran was
dried overnight in vacuo. The dried Dextran was re-dissolved in
de-ionized water (DI-H.sub.2O). The deprotection of Boc group was
performed first by adding HCl for 1 hour to cleave the amide bond
between the Boc group and the protected amine moiety. Subsequently,
TEA was added to increase the pH of the solution up to 9 to
deprotonate the NH.sub.3.sup.+ end groups which were deprotected.
The mixture was then washed twice using methanol and dried in
vacuo. An NMR sample of the dried product was prepared in D.sub.2O
( ). The amine terminated Dextran and carboxyl terminated PLA20
(Mw.about.20 kDa, 6 g, 0.3 mmol) were dissolved in DMSO. The
conjugation between the two polymers was facilitated by adding
catalysts EDC (120 mg, 0.773 mmol) and Sulfo-NHS (300 mg, 1.38
mmol) and allowing reaction to proceed for 4 hours at room
temperature. The resulting Dex-b-PLA was twice precipitated and
purified using excess methanol. In order to remove free Dextran,
the mixture was dissolved in acetone (30 mL) to form a cloudy
suspension. This was centrifuged at 4000 rpm for 10 minutes and the
supernatant was extracted carefully. The supernatant was purged
with air to remove the solvent and then dried overnight in vacuo to
obtain the final copolymers.
[0242] To functionalize the polymers, Dex-b-PLA may be dissolved in
DMSO (30 mg/ml), and added slowly into water under mild stirring.
Periodate oxidation of the Dextran surface was carried out by
adding 60 mg of NaIO.sub.4 and stirring for an hour. Subsequently,
glycerol was added to quench the unreacted NaIO.sub.4. Various
amounts of PBA (i.e. 40 mg for Dex-b-PLA.sub.--40PBA) were added to
the mixture, along with NaCNBH.sub.3, for 24 hours. All reactions
were carried out in the dark. The mixture was then dialyzed in
water for 24 hrs to remove any unreacted solutes, through changing
the wash medium 4 times.
[0243] The polymers or macromolecules described herein may be
formed into a nanoparticle using techniques know to those skilled
in the art, including those discussed in detail below. The geometry
formed by the particle from the polymer or macromolecule may depend
on factors such as the polymers that form the particle. In
addition, also as discussed below, in some cases, the particle may
include a hydrophilic agent or a hydrophobic agent of interest,
depending on the structure of the particle. For example, the
particle may contain a drug or other therapeutic agent. The
hydrophilic or hydrophobic agent may be incorporated in the
particle during formation of the particle, e.g., by including the
agent in a solution containing the polymers that are used to form
the particle, and/or the agent may be incorporated in the particle
after its formation.
[0244] The Dex-b-PLA NPs were prepared using nanoprecipitation
method: 1 mL of Dex-b-PLA in DMSO (10 mg/mL) was added in a
drop-wise manner to 10 mL of DI-H.sub.2O under constant stirring in
order to form NPs. This was stirred for 30 minutes and then dynamic
light scattering (DLS) samples were prepared by extracting 3 mL
samples into polystyrene cuvettes. The sizes of the NPs were
analyzed using 90Plus Particle Size Analyzer (Brookhaven,
.lamda.=659 nm at 90.degree.). The volume averaged multimode size
distribution (MSD) mean diameters were used from the results.
[0245] In addition, the method may employ additional polymers or
macromolecules, which may be distinguishable from the polymers or
macromolecules discussed above. As previously discussed, the first
and second macromolecules may be combined together at different
ratios to produce particles comprising the first and second
macromolecules.
[0246] In some cases, the method may include conjugation with more
than one type of targeting moiety. The surface density of targeting
moiety on the resulting nanoparticles may be controlled by
adjusting the amount of material in the reaction mixture.
[0247] Alternatively, the reaction may occur as a single-step
reaction, i.e., the conjugation is performed without using
intermediates such as N-hydroxysuccinimide or a maleimide, such as
that described in U.S. Pat. No. 8,323,698. However, such method
results in a nanoparticle having a portion of the targeting moiety
located in the core of the particle. Thus, typically, a multi-step
approach will be used to achieve higher targeting efficiently.
[0248] Specific reaction conditions can be determined by those of
ordinary skill in the art using no more than routine
experimentation.
Particular Embodiments
[0249] In some embodiments, there is provided a nanoparticle
composition useful for delivery of a payload to a mucosal site, the
nanoparticle comprising a plurality of amphiphilic macromolecules,
the macromolecules comprising: a hydrophobic portion comprising a
biocompatible polymer selected from a from polylactide, a
polyglycolide, poly(lactide-co-glycolide),
poly(.epsilon.-caprolactone), or a combination thereof; a
hydrophilic portion comprising a biocompatible polymer selected
from polysaccharide, polynucleotide, polypeptide, or a combination
thereof, the hydrophilic portion comprising multiple functional
moieties; and a mucosal targeting moiety selected from a
phenylboronic acid (PBA) derivative, a thiol derivative or an
acrylate derivative, wherein at least a portion of said functional
moieties of the hydrophilic portion are conjugated to the mucosal
targeting moiety.
[0250] In some embodiments, there is provided a nanoparticle
composition useful for delivery of a payload to a mucosal site, the
nanoparticle comprising a plurality of amphiphilic macromolecules,
the macromolecules each comprising: a hydrophobic biocompatible
polymer selected from a from polylactide, a polyglycolide,
poly(lactide-co-glycolide), poly(.epsilon.-caprolactone), or a
combination thereof, the hydrophobic polymer forming the core of
the nanoparticle; a hydrophilic biocompatible polymer selected from
polysaccharide, polynucleotide, polypeptide, or a combination
thereof, having multiple functional moieties, the hydrophilic
portion forming the shell of the nanoparticle; at least a portion
of the functional moieties being conjugated to a mucosal targeting
moiety selected from a phenylboronic acid (PBA) derivative, a thiol
derivative or an acrylate derivative.
[0251] In some embodiments, there is provided a nanoparticle
composition useful for delivery of a payload to a mucosal site, the
nanoparticle comprising a plurality of amphiphilic macromolecules,
the macromolecules comprising: a hydrophobic portion comprising a
polylactide; a hydrophilic portion having multiple functional
moieties, said hydrophilic portion comprising dextran; and a
mucosal targeting moiety being a phenylboronic acid (PBA)
derivative, wherein at least a portion of said functional moieties
of the hydrophilic portion are conjugated to the mucosal targeting
moiety.
[0252] In some embodiments, there is provided a nanoparticle
composition useful for delivery of a payload to a mucosal site, the
nanoparticle comprising a plurality of amphiphilic macromolecules,
the macromolecules each comprising a hydrophobic polylactide
polymer conjugated to a hydrophilic dextran polymer having multiple
functional moieties, at least a portion of said functional moieties
being conjugated to a phenylboronic acid (PBA) derivative.
[0253] In some embodiments, the macromolecule is Dextan-p-PLA. In
some embodiment, the functionalized macromolecule is
Dextran-p-PLA_PBA.
[0254] In some embodiments, the nanoparticle is formed by
conjugating the polylactide to the dextran to form macromolecule,
then forming a nanoparticle, and subsequently
surface-functionalizing the nanoparticle by conjugating at least a
portion of the functional moieties of the dextran to the PBA
derivative to achieve a desired surface density of the PBA
derivative.
[0255] All definitions, as defined and used herein, should be
understood to control over dictionary definitions, definitions in
documents incorporated by reference, and/or ordinary meanings of
the defined terms.
[0256] The indefinite articles "a" and "an," as used herein in the
specification and in the claims, unless clearly indicated to the
contrary, should be understood to mean "at least one."
[0257] In the claims, as well as in the specification above, all
transitional phrases such as "comprising," "including," "carrying,"
"having," "containing," "involving," "holding," "formed from",
"composed of," and the like are to be understood to be open-ended,
i.e., to mean including but not limited to.
[0258] Only the transitional phrases "consisting of" and
"consisting essentially of" shall be closed or semi-closed
transitional phrases, respectively.
[0259] The following examples are intended to illustrate certain
exemplary embodiments of the present disclosure. However, the scope
of the present disclosure is not limited to the following
examples.
EXAMPLES
Example 1
Synthesis and Characterisation of Dex-b-PLA
1.1 Materials
[0260] Acid-terminated poly(D, L-lactide) (PLA, M.sub.w.about.10,
20 and 50 kDa) and PLGA-PEG (PLGA M.sub.w.about.40 kDa, PEG
M.sub.w.about.6 kDa) were purchased from Lakeshore Biomaterials
(Birmingham, Ala., USA). PLA was purified by dissolving in dimethyl
sulfoxide (DMSO) and precipitating in methanol to remove residual
monomers. Dextran (Dex, M.sub.r.about.1.5, 6, and 10 kDa),
hydrochloric acid (HCl), triethylamine (TEA),
N-(3-dimethylaminopropyl)-N-ethylcarbodiimide (EDC), and sodium
cyanoborohydride (NaCNBH.sub.3) were purchased from Sigma Aldrich
(Oakville, ON, Canada), and used without further purification.
N-Hydroxysulfosuccinimide (Sulfo-NHS) and N-Boc-ethylenediamine
were purchased from CNH Technologies (Massachusetts, USA).
Doxorubicin-HCl (MW=580 Da, Intatrade GmBH, Bitterfield, Germany)
was deprotonated by adding TEA (2M equivalent) in the aqueous
solution of Doxorubicin-HCl, and the hydrophobic form of
Doxorubicin was extracted using Dichloromethane (DCM) (Chittasupho,
2009). Borate buffer was prepared at a concentration of 0.05M with
pH of 8.2 by mixing boric acid and sodium hydroxide. Whole sheep
blood (in Alsever's) was purchased from Cedarlane (Burlington, ON,
Canada). Veronal Buffer solution (VBS, 5.times.) was purchased from
Lonza Walkersville Inc (Walkersville, Md., USA). Tritium
[.sup.3H]-PLA-radiolabeled nanocrystals were purchased from
PerkinElmer (Boston, Mass., USA).
1.2 Synthesis of Dex-b-PLA
[0261] The synthesis of the linear block copolymer is divided into
three stages: 1) reductive amination between Dextran and
N-Boc-ethylenediamine, 2) deprotection of the Boc group, and 3)
conjugation of the end modified Dextran with PLA (Scheme 1). The
first step of the synthesis involves reductive amination between
the aldehyde on the reducing end of Dextran and the amine group of
N-Boc-ethylenediamine cross-linker. In a typical reaction, Dex6
(M.sub.r.about.6 kDa, 6 g, 1 mmol) was dissolved in 15 mL of borate
buffer (0.05 M, pH 8.2) with 4 g (2.5 mmol) of
N-Boc-ethylenediamine. The reducing agent, NaCNBH.sub.3 (1 g, 15
mmol), was added to the borate buffer solution and the mixture was
stirred for 72 hours in dark conditions at room temperature. The
mixture was then washed in methanol to remove any unreacted
molecules or catalysts. The end-modified Dextran was dried
overnight in vacuo. H NMR samples were prepared by dissolving the
end-modified Dextran in D.sub.2O (30 mg/mL). The dried Dextran was
re-dissolved in de-ionized water (DI-H.sub.2O). The deprotection of
Boc group was performed first by adding HCl (.about.4 M) for 1 hour
to cleave the amide bond between the Boc group and the protected
amine moiety. Subsequently, TEA was added to increase the pH of the
solution up to 9 to deprotonate the NH.sub.3.sup.+ end groups which
were deprotected. The mixture was then washed twice using methanol
and dried in vacuo. An NMR sample of the dried product was prepared
in D.sub.2O (30 mg/mL). The amine terminated Dextran and carboxyl
terminated PLA20 (Mw.about.20 kDa, 6 g, 0.3 mmol) were dissolved in
DMSO. The conjugation between the two polymers was facilitated by
adding catalysts EDC (120 mg, 0.773 mmol) and Sulfo-NHS (300 mg,
1.38 mmol) and allowing reaction to proceed for 4 hours at room
temperature. The resulting Dex-b-PLA was twice precipitated and
purified using excess methanol. In order to remove free Dextran,
the mixture was dissolved in acetone (30 mL) to form a cloudy
suspension. This was centrifuged at 4000 rpm for 10 minutes and the
supernatant was extracted carefully. The supernatant was purged
with air to remove the solvent and then dried overnight in vacuo to
obtain the final copolymers. NMR samples were prepared at a
concentration of 30 mg/mL in DMSO-d6 for proton NMR and 150 mg/mL
in DMSO-d6 for carbon NMR.
##STR00001## ##STR00002##
1.3 Characterization of Dex-b-PLA Using Nuclear Magnetic Resonance
(NMR)
[0262] The various stages of Dex-b-PLA synthesis were verified
using H NMR spectroscopy (Bruker 300 MHz). The final polymer
conjugation was also verified using C NMR spectroscopy (Bruker 300
MHz). Before any modification, Dextran was dissolved in D.sub.2O
(30 mg/mL) and acid terminated PLA was dissolved in CDCl.sub.3 (5
mg/mL) for preparing NMR samples. As mentioned in the previous
synthesis methods, the end products from the first two steps were
dissolved in D.sub.2O, whereas the final product, Dex-b-PLA, was
dissolved in DMSO-d6 for the NMR analysis.
1.4 Dex-b-PLA NP Formation by Nanoprecipitation
[0263] The Dex-b-PLA NPs were prepared using nanoprecipitation
method: 1 mL of Dex-b-PLA in DMSO (10 mg/mL) was added in a
drop-wise manner to 10 mL of DI-H.sub.2O under constant stirring in
order to form NPs. This was stirred for 30 minutes and then dynamic
light scattering (DLS) samples were prepared by extracting 3 mL
samples into polystyrene cuvettes. The sizes of the NPs were
analyzed using 90Plus Particle Size Analyzer (Brookhaven,
.lamda.=659 nm at 90.degree.). The volume averaged multimode size
distribution (MSD) mean diameters were used from the results.
1.5 Transmission Electron Microscopy
[0264] The particle size and the morphology of the Dex-b-PLA NPs
were further verified using Transmission Electron Microscopy (TEM,
Philips CM10) with the accelerating voltage of 60 kV and the
Lanthanum Hexaboride filament (LaB6). 300 Mesh Formvar coated
copper grids (Canemco & Marivac) were used for this experiment.
The NP suspension in water was prepared using the nanoprecipitation
method as mentioned above. A drop of the NP suspension was placed
onto the grid, and the grid was briefly stained with aqueous
phosphotungstic acid solution. The copper grid with the NP
suspension was dried under ambient environment overnight before
imaging under TEM.
1.6 Results and Discussion
[0265] The synthesis of Dex-b-PLA block copolymers was analyzed
using H NMR spectrometer. As shown in FIG. 1a I, the 4.86 ppm
multiplet was assigned to the proton on carbon 1 of Dextran
repeating units. The 3.14 ppm multiplet was assigned to the proton
on carbon 5 of the non-reducing end the integral ratio between
these two multiplets was used to confirm the MW of Dextran. The
reductive amination reaction of Dextran and N-Boc-ethylenediamine
was confirmed by the presence of 1.3 ppm peak (Boc group) after
removing unreacted free N-Boc-ethylenediamine (FIG. 1a II). The
subsequent deprotection of Boc group exposing the --NH.sub.2
end-group on Dextran was verified by the disappearance of the 1.3
ppm peak (FIG. 1a III). It was shown that the 1.3 ppm peaks were
completely removed after the deprotection steps using HCl and TEA.
After the conjugation of the --NH.sub.2 terminated Dextran with
COOH-terminated PLA (FIG. 1a IV), the excess free Dextran molecules
were removed by precipitating in acetone. The final product shows
peaks corresponding to both the Dextran (multiplets at 4.86 ppm)
and the PLA (multiplets at 5.2 ppm) which confirm the conjugation
of the two polymers (FIG. 1a V). The linear end-to-end conjugation
of PLA and Dextran was also confirmed by Carbon NMR (FIG. 1b). The
peak at 166.81 ppm is assigned to the carbon on PLA that attaches
to the amine terminal of the ethylenediamine linker, while 169 ppm
peak is the carbonyl carbon on PLA backbone (FIG. 1b).
[0266] The size and morphology of NPs using nine formulations of
Dex-b-PLA block copolymers are shown in FIG. 2. Varying the MW of
PLA and Dextran resulted in creating NPs with different sizes
ranging from 15 to 70 nm. As shown in FIG. 2a, increasing the MW of
PLA increased the particle size whereas increasing the MW of
Dextran decreased the particle size. The NP core, formed by PLA,
was predicted to increase in size with increasing MW's of the PLA
chains as demonstrated previously (Riley 1999; Riley 2001) and it
was confirmed here by the NPs composed of PLA MW of 10 kDa, 20 kDa
and 50 kDa. We postulate that the effect of Dextran MW on NP size
is likely due to Dextran configuration on the NP surface. Zahr et
al. found that hydrophilic chains, such as PEG, at MW of 5 kDa or
longer would be able to "fold-down" onto the particle surface
creating a mushroom conformation (Zahr 2006). Similarly, this
phenomenon may explain why the NPs with longer Dextran chains lead
to smaller hydrodynamic diameters. The shorter Dextran chain length
has a smaller degree of freedom and confined to linear structure
compared to those with longer chain length. The TEM image of NPs
composed of PLA20-Dex6 (MW.sub.PLA.about.20 kDa,
MW.sub.Dextran.about.6 kDa) confirmed the particles exhibit
spherical structure (FIG. 2b).
[0267] Dex-b-PLA NPs with sizes under 50 nm have been synthesized
using the simple process of bulk nanoprecipitation. PLGA-PEG block
copolymer was used as a commercial benchmark, which formed NPs with
size 133.9.+-.6.1 nm following the same procedure. The particle
size for PLGA-PEG is in agreement to previous literature values
(Dhar 2009). PLGA-PEG NPs have been able to achieve smaller
particle sizes but it required the assistance of microfluidic
devices for enhanced control (Karnik 2008). The particle size of
Dex-b-PLA NPs, on the other hand, can be controlled simply by
changing the MW of the compositional polymers as exemplified in
FIG. 2a.
Example 2
Encapsulation and In Vitro of Doxorubicin in Dex-b-PLA NPs Via
Nanoprecipitation
[0268] The encapsulation of Doxorubicin in the Dex-b-PLA NPs was
accomplished using nanoprecipitation method. Dex-b-PLA and
Doxorubicin were both dissolved in DMSO (Dex-b-PLA concentration of
7 mg/mL, with varying drug concentrations). 1 mL of the DMSO
solution is added drop-wise into 10 mL of water under stirring and
continued to stir for additional 30 minutes. The NPs in water were
filtered through syringe filter (pore size=200 nm) to remove the
drug aggregates and subsequently filtered through Amicon filtration
tubes (MWCO=10 kDa, Millipore) to further remove any remaining free
drugs in the suspension. The filtered NPs containing encapsulated
Doxorubicin were resuspended and diluted in DMSO. Consequently, the
drug loading (wt %) in the polymer matrix was calculated by
measuring concentration of the Doxorubicin in the mixture by
obtaining the absorbance of the solution at 480 nm using Epoch
Multi-Volume Spectrophotometer System (Biotek). The measurements
were obtained in triplicates (n=3, mean.+-.S.D). The absorbance
measured from same procedure using the polymers without the drugs
was used as the baseline. The absorbance was correlated with the
concentration of the Doxorubicin in DMSO by using standard
calibration obtained. The same procedure was used for PLGA-PEG to
encapsulate Doxorubicin for comparative analysis. The encapsulation
efficiency (%) and drug loading (wt %) were calculated using the
two equations (Eq. 1 and Eq. 2).
Encapsulation efficiency ( % ) = mass of drug encapsulated mass of
initial drug feed .times. 100 % ( 1 ) Drug loading ( wt % ) = mass
of drug encapsulated mass of the nanoparticle .times. 100 % ( 2 )
##EQU00001##
[0269] Based on the size tuning as shown in FIG. 2, PLA20-Dex10
(MWPLA.about.20 kDa, MWDextran.about.10 kDa) and PLA20-Dex6 were
selected for analyzing the encapsulation efficiencies and the drug
loading using Doxorubicin as a model hydrophobic drug (FIG. 3). The
particle sizes for PLA20-Dex10 and PLA20-Dex6 were 20.5 and 30.1 nm
respectively. Doxorubicin compounds were incorporated into NPs
through nanoprecipitation method. Both Dextran based NPs,
PLA20-Dex10 and PLA20-Dex6 NPs, were found to encapsulate large
amounts of Doxorubicin with maximum loadings of 21.2 and 10.5 wt %
respectively. The maximum loadings were achieved at 40 wt % initial
loading, and further increase in the initial loading did not
increase the drug loading in the NPs due to aggregation of the
particles. It is speculated that PLA20-Dex10, with longer Dextran
chain than PLA20-Dex6, is likely to have more Doxorubicin weakly
associated on the NP surface or encapsulated near the surface of
the NPs during nanoprecipitation. This effect was minimized by
conducting ultrafiltration (MWCO=10 kDa) after the
nanoprecipitation ensuring that the non-specifically bound drugs
were removed from the NP suspension. The maximum drug loading in
PLGA-PEG NPs, used as a control, was found to be 7.1 wt %. It was
found that excess initial loading caused more drug precipitation
and particle aggregation during nanoprecipitation for PLGA-PEG NPs,
whereas Dex-b-PLA NPs showed negligible size increases even at
their maximum drug loading. The maximum Doxorubicin loading
achieved with PLA20-Dex10 NPs were considerably higher than the
most reported values using PEG based NPs in the literatures, which
varied over 4.3-11.2% for poly(.epsilon.-caprolactone)-PEG
copolymers (Shuai 2004; He 2010), 8.7% for poloxamer 407 and PEG
hydrogel system (Missirlis 2006), and 18% for
PEG-poly(3-benzyl-L-aspartate) based NPs (Kataoka 2001). The
increased drug loading is most likely due to the greater
hydrophilicity of Dextran compared to PEG (Alpert 1990), which in
turn reduces the probability of Dextran chains from the block
copolymers associating in the hydrophobic core of the NPs. The
encapsulation efficiency and the total drug payload using the
Dex-b-PLA system is comparable to commercially available liposomal
systems such as the FDA approved Doxil.RTM., which has a drug
loading of 12.5% and DaunoXome.RTM., which has Daunorubicin loading
of 7.9% (Drummond 1999). The concentration of Doxorubicin in the
Doxil.RTM. formulation translates into 6.25 mg/m.sup.2 when
Doxil.RTM. is administered at 50 mg/m.sup.2 (Drummond 1999; Safra
2000). The same physiological concentration of Doxorubicin can
theoretically be achieved using only 30 mg/m.sup.2 of PLA20-Dex10
NP-Doxorubicin formulation.
Example 3
In Vitro Release of Doxorubicin from Dex-b-PLA NPs
[0270] Using the procedure described in the previous example, drug
encapsulated NPs were prepared and filtered to remove
non-encapsulated drug aggregates. A purified sample of NPs-drug
suspension was collected to measure the maximum absorbance and this
was used as the 100% release point. Subsequently, the NP-drug
suspension was injected into a Slide-a-Lyzer Dialysis cassette
(MWCO=20 kDa, Fisher Scientific) and dialyzed against 200 mL of
phosphate buffered saline (PBS, pH 7.4) at 37.degree. C. under mild
stirring. At predetermined time intervals, 1 mL of the release
medium was extracted and the same volume of fresh new PBS was added
to the release medium. The extracted release medium was used to
perform UV-Vis absorption measurements at 480 nm in triplicates
(n=3, mean.+-.S.D). The release medium was replaced several times
to maintain the concentration of Doxorubicin in the medium below 3
.mu.g/mL and to stay below the solubility limit of the Doxorubicin
in PBS. Replacing the medium was also expected to prevent the
adhesion of released Doxorubicin to the glass walls of the beaker
or the magnetic stir bar. The release of Doxorubicin from PLGA-PEG
was also obtained with identical procedure for comparative
analysis. Free Doxorubicin, without any polymers, release was also
observed using the same procedure and all three release profiles
from the NPs were normalized using the free Doxorubicin release
data along with encapsulation efficiency data. This normalization
resulted in a release curve for only encapsulated Doxorubicin. All
experiments were performed in dark environment, and the beakers
were sealed with Parafilm to prevent evaporation of PBS.
[0271] The in vitro release of Doxorubicin from the NPs was carried
out in pH 7.4 PBS buffer at 37.degree. C. As shown in FIG. 4, the
release profile of Doxorubicin from NPs was characterized with an
initial burst followed by a sustained-release phase. It is possible
that the burst-release region corresponds to drugs non-specifically
bound on the surface of the NPs, or drugs encapsulated near the
surface of the NPs during the nanoprecipitation procedure
(Magenheim 1993). PLA20-Dex6 and PLA20-Dex10 NPs exhibited
burst-release region within the initial 24 hours, releasing up to
48% and 74% respectively. The subsequent sustained-release phase of
Doxorubicin from PLA20-Dex6 and PLA20-Dex10 NPs continued for 192
hours with similar rate of release from both NPs. The
sustained-release phase may correspond to the diffusional release
of the drugs from the core of the NPs. In the control study using
PLGA-PEG NPs, the burst-release phase of Doxorubicin was within the
first 6 hours while steady-release phase continued up to 96 hours,
similar to what has been reported previously (Esmaeili 2008).
Example 4
Hemolysis Assay
[0272] Dex-b-PLA NPs were purified by using Amicon filtration tubes
(MWCO=10 kDa) and centrifugation at 4100 rpm for 30 minutes. A
concentration range of NPs was obtained by this process. These NPs
were then incubated at 37.degree. C. for one hour with 200 .mu.L of
sheep erythrocytes with red blood cells concentration of
1.times.10.sup.8 cells/mL to obtain a final volume of 1 mL per
sample. The percent hemolysis was calculated by measuring the
absorbance at 415 nm and using the absorbance at 500 nm as the
baseline. The measurements were conducted in triplicates
(mean.+-.S.D). Here, VBS solution was used as the negative control
and deionized water was used as the positive control. PLGA-PEG NPs
were also prepared and tested in a similar manner for
comparison.
[0273] Previous work has considered hemolysis of NPs less than 5%
to be biocompatible (Dobrovoiskaia 2008). It has been demonstrated
that PLGA NPs stabilized by surfactants are severely hemolytic to
80% and hemolysis is reduced considerably by using a hydrophilic
PEG surface in the case of PLGA-PEG NPs (Kim 2005). The same
results were expected from the use of Dextran based NP formulation
since Dextran derivatives such as diethylaminoethyl-dextran have
low (.about.5%) hemolysis (Fischer 2003). The block copolymer NPs
formulated previously were tested for hemolytic activity at various
concentrations (1-10 mg/mL). It was shown that all formulated NPs
were not significantly hemolytic (<5%) up to a concentration of
10 mg/mL in the blood (FIG. 5). The hemolysis by both PLA20-Dex6
and PLA20-Dex 10 were similar since they have the same component
polymers. For comparison, Doxil.RTM. (a liposomal formulation of
doxorubicin) is usually administered at the dose of 50 mg/m.sup.2
(Safra 2000). This dose translates to a concentration of 0.018
mg/mL in blood for an average human being (body surface area 1.79
m.sup.2 (Sacco 2010), and blood volume 5 L) (Kusnierz-Glaz 1997).
The tested hemocompatible concentration (10 mg/mL) for PLA-b-Dex
NPs is considerably higher than the administered dose of
Doxil.RTM.. This suggests that PLA-b-Dex NPs are a safe system for
intravenous administration.
Example 5
Pharmacokinetics and Biodistribution of Dex-b-PLA NPs
[0274] To ensure that all radioactivity administered to rats was
associated with the particles, tritium [.sup.3H]-PLA-radiolabeled
nanocrystals were washed and purified in methanol prior to NP
formation. Albino Wistar rats, body weight between 200 and 250 g,
were fasted overnight but had free access to water. 200 .mu.L of
the NP formulations were prepared in NaCl 0.9% and injected
intravenously into the tail vein at a dose of approximately 30
mg/kg. Blood (approximately 200 .mu.L) was collected in heparinized
microcentrifuge tubes by controlled bleeding of hind leg saphenous
veins at the indicated time intervals. To characterize the
biodistribution of NPs, rats were euthanized at 24 h after NP
injections. Approximately 200 .mu.L of blood was drawn by cardiac
puncture from each mouse. Organs including heart, lungs, liver,
spleen and kidneys were harvested from each animal as described
previously (Gu 2008). The .sup.3H content in the tissue and blood
were assayed in a Wallac 1414 Liquid Scintillation Counter.
[0275] The NP circulation half-life in vivo was characterized by
measuring the amount of tritium [.sup.3H]-PLA-radiolabeled
nanocrystals that were incorporated in the NP formulations. FIG. 6
shows NP concentration in blood circulation at predetermined time
intervals after intravenous administration. It is noted that the
time-dependent NP concentration in the blood were characterized by
two regions of distinct slopes. The first region (first .about.18
hrs) corresponds to the initial clearance of the NPs from the blood
circulation, whereas the second region indicates the terminal
clearance of the NPs. The former region profiles the NP volume of
distribution among vascular and extravascular tissues, while the
terminal half-life relates to the systemic clearance of the NPs
from the body (Yang 2009). The initial half-life (t.sub.1/2),
terminal half-life t.sub.z1/2), the blood retention time for 90% of
the NPs (t.sub.0.9), and AUC (Gaucher 2009) of the three NPs are
summarized in Table 1. At 24 hours postinjection, rats were
euthanized, and the major organs were harvested from the animals to
evaluate the biodistribution of the NPs (FIG. 7). It was observed
that all three NPs had maximum accumulation in the liver and the
percent distribution was similar for each NP. Higher accumulations
in the spleen were observed with PLGA-PEG NPs compared to both of
Dex-b-PLA NPs (p<0.01). Accumulation of NPs in all other organs
was below 5% with similar amount of accumulation among the NPs in
each organ.
[0276] Although all three types of NPs showed similar t.sub.z1/2
values, both PLA20-Dex10 and PLA20-Dex6 NPs showed significantly
higher values of t.sub.1/2, t.sub.0.9, and AUC compared to that of
the model NPs composed of PLGA-PEG. Previous studies mainly focused
on t values for NPs but the present inventors extracted t.sub.0.9
values for comparison purposes. It was observed that t.sub.0.9
values were only about 2 hrs for PEG-b-PLA NPs (Gaucher 2009), 6
hrs for polyvinylpyrrolidone based NPs (Gaur 2000) and about 8 hrs
for chitosan based NPs (He 2010). Not only do Dex-b-PLA NPs
outperform these NPs with a t.sub.0.9 of 38.3 hrs, they are also
comparable to 60 nm PEG-b-PCL system (Lee 2010) and Stealth.RTM.
liposomes (Allen 1991), both of which have t.sub.0.9 values over 48
hours. In this study, the longer blood circulation observed in
Dex-b-PLA NPs, compared to PLGA-PEG NPs, is believed to be
partially due to the size difference. A recent study by Rehor et
al. showed that NPs with diameter of 40 nm had longer circulation
half-life compared to larger NPs with diameter of 100 nm (Rehor
2008). It is hypothesized that Dex-b-PLA NPs, having smaller sizes
than PLGA-PEG NPs, have increased curvature that reduce protein
adsorption, which may in turn result in slower clearance rate by
the RES. This is further supported by the longer blood circulation
time of PLA20-Dex10 compared PLA20-Dex6 since the former has
smaller particle size. In addition to their size effect on protein
adsorption, it is also hypothesized that the abundant hydroxyl
groups on the Dextran surface may induce sufficient hydration layer
around the NPs to limit protein adsorption (Portet 2001). It has
been reported that accumulation rate in tissues such as spleen
increases with increase in the particle sizes (Li 2008) which is
consistent with the current findings. It has also been observed
that PEG coating in PEGylated particles can increase accumulation
in the spleen (Peraccia 1999) whereas the neutrality (Chouly 1996)
and flexibility (Passirani 1998) of dextran chains on the NP
surface can cause lower protein absorption leading to lower spleen
accumulation. Dex-b-PLA NPs are expected to have low complement
activation as observed for dextran-poly(methyl methacrylate) NPs,
whose behaviour was similar to soluble dextran (Passirani 1998).
The lower accumulation of the Dex-b-PLA NPs in spleen along with
lower complement activation may have attributed to their longer
blood circulation (Meerasa 2011). The long circulation half-life of
NP drug carriers is a crucial parameter in cancer therapy since it
increases the probability of accumulating at cancerous tissues due
to EPR effect: particle size below 100 nm directly promotes
accumulation of NPs in the tumor sites since the vascular pores
around tumor are at least 100 nm in size (Cho 2008). The
size-tuneable Dex-b-PLA system developed here presents a polymeric
platform for systematically studying the effect of NP size on
various in vivo characteristics such as biocompatibility, blood
clearance, tumor accumulation and biodistribution and screening
candidates for further clinical evaluation.
TABLE-US-00001 TABLE 1 Blood pharmacokinetic parameters for
PLA20-Dex10, PLA20-Dex6, and PLGA-PEG NPs t.sub.1/2 (hr) t.sub.z1/2
(hr) t.sub.0.9 (hr) AUC PLA20-Dex10 12.3 .+-. 2.2 29.8 .+-. 1.0
38.3 .+-. 21.5 1040 PLA20-Dex6 7.2 .+-. 0.4 26.6 .+-. 3.1 17.9 .+-.
8.6 691 PLGA-PEG 3.7 .+-. 0.6 27.0 .+-. 2.3 5.0 .+-. 2.4 287
t.sub.1/2: initial half-life; t.sub.z1/2: terminal half-life;
t.sub.0.9: blood retention time for 90% of the NPs; AUC: Area under
curve (% dose hr)
[0277] Statistical analysis was performed using the student t-test
and statistical significance was assessed with p<0.01.
Example 6
Synthesis and Characterization of Dex-b-PLA-BLA NPs
6.1 Synthesis of Dex-b-PLA
[0278] The synthesis of Dex-b-PLA was carried out as described in
Example 1 (see also, Verma, 2012). Briefly the procedure for the
synthesis of Dex-b-PLA divides into three stages: reductive
amination between Dextran and N-Boc-ethylenediamine, deprotection
of the Boc group, and conjugation of the amine-modified Dextran end
group with carboxyl-terminated PLA. Reductive amination was carried
out by dissolving Dex in borate buffer and mixing it with
N-Boc-ethylenediamine and NaCNBH.sub.3 in dark condition for 72
hrs. After the reaction the mixture is washed with methanol and
dried in vacuum desiccator. The sample is then dissolved in
DI-H.sub.2O and treated with hydrochloric acid and triethyl amine
for the deprotection of the Boc group. The conjugation of
amine-terminated Dextran and PLA was carried out in DMSO with EDC
and Sulfo-NHS as catalysts for 4 hrs. The final product was washed
several times with methanol. The wash sample was further dissolved
in acetone and centrifuged. The supernatant was extracted carefully
in order to separate from free unreacted Dextran that have been
precipitated. Finally the supernatant containing Dex-b-PLA was
dried in vacuum desiccator.
6.2 Surface Functionalization of Dex-b-PLA NPs with PBA
[0279] Dex-b-PLA was dissolved in DMSO (30 mg/ml), and added slowly
into water under mild stirring. Periodate oxidation of the Dextran
surface was carried out by adding 60 mg of NaIO.sub.4 and stirring
for an hour. Subsequently, glycerol was added to quench the
unreacted NaIO.sub.4. Various amounts of PBA (i.e. 40 mg for
Dex-b-PLA.sub.--40PBA) were added to the mixture, along with
NaCNBH.sub.3, for 24 hours. All reactions were carried out in the
dark. The mixture was then dialyzed in water for 24 hrs to remove
any unreacted solutes, through changing the wash medium 4
times.
6.3 Characterization of Dex-b-PLA_PBA NPs
[0280] The surface modification with PBA was verified using .sup.1H
NMR spectroscopy (Bruker 300 MHz). Dex-b-PLA_PBA polymers were
dissolved in DMSO-d6 (25 mg/ml) for the .sup.1H NMR
characterization. UV-Vis absorption measurement at 291 nm was
performed with Epoch Multi-Volume Spectrophotometer System (Biotek,
USA) on the Dex-b-PLA_PBA in order to quantify the amount of PBA
attached to the Dextran chains. Dex-b-PLA solution with same
concentration was used as the baseline for UV-vis absorption study.
The NPs of Dex-b-PLA_PBA prepared using nanoprecipitation were also
analyzed using 90Plus Particle Size Analyzer (Brookhaven,
.lamda.=659 nm at 90.degree.) obtaining volume-averaged multimode
size distribution (MSD) mean diameter. The particle size and
morphology of Dex-b-PLA_PBA NPs were further confirmed verified
using TEM (Philips CM10) with the accelerating voltage of 60 kV and
the Lanthanum Hexaboride filament (LaB6). 300 Mesh Formvar coated
copper grids (Canemco & Marivac) were used for this experiment.
The NP suspension in water was prepared using the nanoprecipitation
method as mentioned above. A drop of the NP suspension was placed
onto the grid, and the grid was briefly stained with aqueous
phosphotungstic acid solution (2 w/v % in water). The copper grid
with the NP suspension was dried under ambient environment
overnight before imaging under TEM.
[0281] The NMR spectrum of Dex-b-PLA_PBA shows peaks corresponding
to both PLA (multiplets at 5.2 ppm) and Dextran (multiplets at 4.86
ppm), while also showing multiplet peaks at 6.6 ppm and 6.9 ppm,
which correspond to the protons from carbon 2 to 6 in the phenyl
group of the PBA (FIG. 12a). UV absorption at 291 nm was measured
to quantify the amount of PBA on the NPs with respect to the
Dextran monomers. Increasing the amount of PBA in the initial
reaction mixture proportionally increased the final PBA conjugation
on the Dextran surface (Table 2) with the highest density of 34.6
mol % (equivalent of about 3.5 PBA conjugated per 10 Dextran
monomers) achieved for Dex-b-PLA.sub.--320PBA NPs.
[0282] Conjugation of Cysteamine onto the Dex-b-PLA NP surface was
also demonstrated using .sup.1H NMR spectrum (FIG. 13). The peaks
that correspond to the protons on carbon 1 and 2 of the cysteamine
are shown as multiplets peaks near 2.7 ppm. However, higher
resolution NMR characterization is required in the future to
further differentiate the peaks from other noise peaks from the
polymer.
[0283] The sizes and morphology of the nanoparticles were analyzed
using the procedure illustrated in Example 1. The sizes of the NPs
were in the range of 25 to 28 nm, which are smaller than the
unmodified NPs of 47.9 nm. Without being bound by theory, it is
postulated that the particle size reduction is attributed by the
PBA molecules causing the Dextran chains to be less hydrophilic,
leading them to form more compact shells around the PLA particle
core. TEM images confirmed a spherical morphology, due to the
formation of a core-shell structure of the amphiphilic block
copolymers (FIG. 12b). The sizes of Dex-b-PLA_PBA NPs obtained are
smaller than that normally achieved with PEG-based block copolymers
such as PLGA-PEG (Karnik, 2008). We postulate that smaller NPs may
be more desirable for mucoadhesion, since they provide greater
surface area for interaction with the mucous membrane.
Example 7
Drug encapsulation in Dex-b-PLA_PBA NPs
[0284] The Cyclosporine A (CycA) encapsulation in the Dex-b-PLA and
Dex-b-PLA_PBA NPs were measured using the procedure described in 0.
Maximum encapsulation of CycA was achieved at an initial feed of 40
wt/wt %: Dex-b-PLA NPs encapsulated up to 10.8 wt/wt %, whereas
Dex-b-PLA.sub.--40PBA and Dex-b-PLA.sub.--320PBA encapsulated up to
11.2 and 13.7 wt/wt %, respectively (FIG. 15). The 13.7 wt/wt %
encapsulation is equivalent to 2.38 .mu.g of CycA in 28 .mu.L
formulation (which is the same as the administration volume of
commercially available RESTASIS.RTM.), whereas the commercial
product contains 14 .mu.g. Therefore, a therapeutically relevant
dosage can be achieved by simply adjusting the polymer and drug
concentration in the formulation.
[0285] Encapsulation of other types of bioactive agents in the
Dex-b-PLA_PBA NPs has also been explored (FIG. 17). In one
embodiment the bioactive agent is Dorzolamide, which is commonly
used to treat glaucoma. In another embodiment the bioactive agent
is Brinzolamide, which is also used to treat glaucoma. Natamycin,
which is an antifungal agent, is also used as a bioactive agent in
the encapsulation in the Dex-b-PLA_NPs. In other embodiments,
Doxorubicn, an anti-cancer agent, and Olopatadine, antihistamine,
were also explored in the encapsulation in the PLA-Dex NPs.
[0286] The encapsulation of Cyclosporine A (CycA) in the Dex-b-PLA
NPs was accomplished using nanoprecipitation method. Dex-b-PLA and
CycA were both dissolved in DMSO (Dex-b-PLA concentration of 7
mg/mL, with varying drug concentrations). 1 mL of the DMSO solution
is added drop-wise into 10 mL of DI-H.sub.2O under mild stirring
and continued to stir for additional 30 minutes. The NPs in water
were filtered through syringe filter (pore size=200 nm) to remove
the drug aggregates and subsequently centrifuged using Amicon
filtration tubes (MWCO=10 kDa, Millipore) to further remove any
remaining free drugs in the suspension. The filtered NPs containing
encapsulated CycA were resuspended and diluted in Acetonitrile.
Consequently, the drug loading (wt/wt %) in the polymer matrix was
calculated by measuring concentration of the CycA in the mixture
using High-performance liquid chromatography (HPLC, Thermo
Scientific). The measurements were obtained in triplicates (n=3,
mean.+-.S.D). The absorbance measured from same procedure using the
polymers without the drugs was used as the baseline. The
measurements were converted to the concentration of the CycA using
standard calibration obtained.
[0287] The encapsulation of Dorzolamide, Brinzolamide, and
Natamycin in the Dex-b-PLA NPs were accomplished using the same
method. The characterization of these drugs was performed using
Multi-Volume Spectrophotometer System (Biotek, USA) instead of
HPLC.
Example 8
Drug Release from Dex-b-PLA_PBA NPs In Vitro
[0288] The in vitro release phenomenon of CycA from the Dex-b-PLA
NPs were analyzed using the procedures described in 0. Both
Dex-b-PLA and Dex-b-PLA_PBA NPs (Dex-b-PLA.sub.--40PBA and
Dex-b-PLA.sub.--320PBA) showed a total release point at around 120
hrs (FIG. 16), which is significantly longer than previous studies
involving in vitro release of CycA from micro or nanoparticles
which showed up to 48 hours of sustained release (Li, 2012; Shen,
2010; Shen, 2010; Yuan, 2006). Moreover, the release rate may
potentially be a significant improvement over the commercial
product, which requires administering twice a day. Whereas
Dex-b-PLA NPs demonstrated a sustained release rate for up to 120
hours, Dex-b-PLA_PBA NPs showed two regions of slightly different
release rate. In the first 48 hrs, the Dex-b-PLA_PBA NPs released
CycA at a faster rate compared to Dex-b-PLA NPs, which may be due
to the release of CycA that were encapsulated near the slightly
more hydrophobic surface Dex-b-PLA_PBA NPs. The subsequent slower
release rate, compared to Dex-b-PLA NPs, may be due to the release
of drugs from the core of the Dex-b-PLA_PBA NPs, which need to
diffuse through the more compact Dextran surface. When the volume
of PBA modified NP formulation were scaled to the administration
volume of RESTASIS.RTM. (28 .mu.L), the CycA release rates were in
the range of .mu.g/day, which is similar to the daily
administration dosage of CycA in RESTASIS.RTM.. Therefore, it is
possible to optimize the formulation by changing the concentration
of the PBA modified NPs and/or the amount of CycA to achieve a
clinically effective release rate and amount.
[0289] In vitro CycA release phenomena from both PBA modified and
unmodified Dex-b-PLA NPs in the STF at 35.degree. C. were analyzed
by quantifying the CycA in the STF at predetermined time intervals
using High Performance Liquid Chromatography (HPLC). Using the
procedure described in the previous section, drug encapsulated NPs,
both Dex-b-PLA and Dex-b-PLA_PBA, were prepared and filtered to
remove non-encapsulated drug aggregates. A purified sample of
NPs-drug suspension was collected to measure the maximum absorbance
and this was used as the 100% release point. Subsequently, the
NP-drug suspension was injected into a Slide-a-Lyzer Dialysis
cassette (Molecular weight cut-off=20 kDa; Fisher Scientific,
Canada) and dialyzed against 200 mL of simulated tear fluid (STF)
at 35.degree. C. under stirring. At predetermined time intervals, 1
mL of the release medium was extracted and the same volume of fresh
new STF was added to the release medium. The extracted release
medium was characterized using HPLC method (n=3, mean.+-.S.D). The
release medium was replaced several times to maintain the
concentration of CycA in the medium in order to stay below its
solubility limit in water. Replacing the medium was also expected
to prevent the adhesion of released CycA to the glass walls of the
beaker or the magnetic stir bar. The beakers were sealed with
Parafilm to prevent evaporation of water.
[0290] The in vitro release study of Dorzolamide, Brinzolamide, and
Natamycin in the Dex-b-PLA NPs were accomplished using the same
method. The characterization of these drugs was performed using
Multi-Volume Spectrophotometer System (Biotek, USA) instead of
HPLC.
Example 9
Mucoadhesion Tests
9.1 In Vitro Mucoadhesion Test--Zeta Potential
[0291] Zetapotential measurements were used to analyze the
interaction between mucin particles and the NPs using the
procedures described in 0. Several reports in the past have used
zeta potential to assess the mucoadhesive properties of drug
carriers (Khutoryanskiy, 2011; Shaikh, 2011; Sogias, 2008; du Toit
2011; Takeuchi, 2005). Mucin particles at physiological pH exhibit
overall negative surface charge due to the presence of carboxylate
groups (sialic acid) and ester sulfates at the terminus of sugar
units (Khutoryanskiy, 2011). By adhering to the sialic acid
moieties of the mucin particles, the Dex-b-PLA_PBA NPs may shield
the negative charges from the surface of the mucin particles and
also cause aggregation of the mucin particles, thus increasing the
overall surface charge. Only Dex-b-PLA.sub.--160PBA (22.9 mol %
PBA) and Dex-b-PLA.sub.--320PBA NPs (34.6 mol %) showed significant
interaction with mucin particles compared to the control study
(Table 2). Low PBA surface functionalization densities do not
appear to show a difference compared to unmodified NPs in terms of
mucin-NP interaction. It is therefore desirable to use NPs with
abundant surface functional groups, such as Dextran-based NPs, to
tune the functionalization density where maximum mucoadhesion is
desired. If one was to functionalize the surface of PLGA-PEG NPs,
using one functional group per each PEG chain, the maximum PBA
modification can be achieved is only 0.44 mol % (assuming the same
MW of PEG, i.e. 10 kDa). An increased amount of PBA
functionalization also increased the extent of NP-mucin
interaction, which allows potential increase of mucin-NP
interaction by saturating PBA on the surface. However, the
functionalization of PBA causes the Dextran surface to be more
hydrophobic, increasing the potential for aggregation of the NPs.
It is therefore ideal to tune the amount of PBA functionalization
to achieve optimal mucin-NP interaction without compromising the NP
colloidal stability.
TABLE-US-00002 TABLE 2 PBA conjugation efficiency and diameter of
unmodified and modified Dex-b-PLA NPs PBA:Dex.sup.a) Diameter Zeta
potential.sup.b) Formulation (mol %) (nm) (mV) Mucin -11.1 .+-. 0.1
Dex-b-PLA 0 47.9 .+-. 0.5 -10.7 .+-. 0.6 Dex-b-PLA_10PBA 2.85 .+-.
0.03 27.5 .+-. 0.9 -11.4 .+-. 0.2 Dex-b-PLA_40PBA 12.2 .+-. 0.2
26.7 .+-. 0.1 -10.8 .+-. 0.4 Dex-b-PLA_160PBA 22.9 .+-. 0.3 25.2
.+-. 1.0 -9.67 .+-. 0.76 Dex-b-PLA_320PBA 34.6 .+-. 0.2 28.1 .+-.
0.3 -8.32 .+-. 0.28 .sup.a)Mol % of PBA with respect to Dextran
monomers; .sup.b)NP suspensions are mixed with mucin suspension in
PBS
[0292] To assess the mucoadhesive properties of PBA modified
Dex-b-PLA, zeta potential was measured for quantitative analysis of
interaction between mucin particles and Dex-b-PLA_PBA NP
suspension. 1 w/v % mucin solution was prepared in pH 7.4 PBS by
stirring overnight and the solution was subsequently sonicated for
10 minutes (Branson Digital Sonifier 450, USA). To 700 .mu.L of
mucin particle solution were added 200 .mu.L of 0.7 mg/ml
Dex-b-PLA_PBA NP suspension in PBS. A control study was also
performed by adding 200 .mu.L of PBS to the mucin particle
solution. The zeta potential of mucin particles with the NP
suspension and the control study were determined using a Malvern
ZetaSizer Nano ZS90 (Malvern Instruments, Worcestershire,
U.K.).
9.2 In Vitro Mucoadhesion Test--PAS Staining Method
[0293] Mucoadhesion of the NPs was measured using the in vitro PAS
staining method as described above. Compared to the Dex-b-PLA and
the PLGA-PEG NPs, the Dex-b-PLA_PBA NPs showed increased mucin
adsorption (Table 2). Dex-b-PLA.sub.--10PBA, Dex-b-PLA.sub.--40PBA,
and Dex-b-PLA.sub.--160PBA NPs showed a linear increase in mucin
adsorption from 0.575 to 0.605 mg/mg of NPs as the degree of PBA
surface functionalization increased. However, further increase in
PBA surface functionalization (i.e. Dex-b-PLA.sub.--320PBA)
decreased the amount of mucin adsorbed. Without wishing to be bound
by theory, it is possible that excess functionalization of the NP
surfaces with PBA causes the Dextran to become more hydrophobic.
This would increase the potential for self-aggregation of the NPs,
reducing the total available surface area for mucin adsorption. It
is therefore ideal to tune the amount of PBA functionalization to
achieve optimal mucin-NP interaction without compromising the NP
colloidal stability. It is also possible that smaller NPs render
higher mucin adsorption due to their larger total surface area, as
shown by comparing PLGA-PEG, Dex-b-PLA, and Dex-b-PLA_PBA NPs.
However, as each type of NP exhibit different surface properties,
the trend is inconclusive. The Dex-b-PLA_PBA NPs all exhibited
significantly higher mucin adsorption compared to the previous
studies involving chitosan based NPs and thiolated NPs, which
showed about 0.25 and 0.13 mg/mg of NPs respectively at 1 hr
incubation (Lee, 2006).
[0294] Mucoadhesion was calculated as the amount of mucin adsorbed
per mg of NPs. NP suspension (1 ml) was mixed with 1 ml of mucin
solution (1 mg/ml in STF) and incubated at 37.degree. C. for 1 hr.
The mixture was then centrifuged at 15,000 rpm for 1 hr and free
mucin in the supernatant was quantified using the periodic
acid/Schiff (PAS) staining method (Lee, 2006). Mucin adsorption was
calculated by subtracting the free mucin concentration from the
initial mucin concentration. Mucin standards (0.1, 0.25 and 0.5
mg/ml) were determined using the same procedure to obtain a
calibration curve.
Example 10
In Vivo Studies
10.1 Acute Response Study Using Dex-b-PLA_PBA NPs
[0295] To analyze the short-term biocompatibility of the NP
formulation, the formulations were administered to rabbit eyes,
while having contralateral eyes as control, and daily slit-lamp
examination for up to 7 days was performed to analyze the ocular
surface. Upon grading of the 7 categories (discomfort, conjunctival
swelling and redness, lid swelling, discharge, corneal
opacification, and infiltrate) from 0 (none) to 4 (severe), the
control eyes showed overall higher values compared to the
corresponding NP treated eyes (FIG. 23). Throughout the duration of
the study, conjunctival swelling, corneal opacification, and
infiltrate were not observed in any of the rabbits.
[0296] Three female rabbits (New Zealand Albino) were used for this
study. The rabbits were acclimated for one week prior to the
experiment. The nanoparticles are prepared using the
nanoprecipitation method described in Example 4 but without the
drug. The nanoparticles were filtered using 200 nm syringe filter,
and further sterilized using UV irradiation inside a BioSafety
Cabinet (BSC) for 1 hour. One eye was administered with NPs (28
.mu.l; 19 .mu.g of Dex-b-PLA_PBA NPs) while the contra-lateral eye
is used as control. Slit lamp examination at 0, 1, 8, 24, 48, 72,
96, 120, 144, and 168 hr after administration was used to evaluate
7 different categories (Note that 0 hr means before
administration). These 7 categories (discomfort, conjunctival
redness and swelling, lid swelling, discharge, corneal
opacification, and infiltrate) were graded from 0 (no sign) to 4
(severe). After 168 hrs, the rabbits were euthanized, and the
ocular tissues were collected in formalin for further
histopathology analysis.
10.2 Histopathology Analysis of Ocular Tissues
[0297] After the duration of slit-lamp examination, the rabbits
were euthanized and the ocular tissues (the entire ocular globe,
and upper and lower eyelids) were collected for histopathology
analysis (0). From examining the cornea, bulbar and tarsal
conjunctivas of all the eyes, normal ocular tissues surfaces were
observed in both the NP treated and the control eyes (FIG. 24). All
eyes showed anterior segment with preserved architecture and
morphology. No sign of inflammation, altered layer integrity, or
presence of residual particles were detected in any of the eyes.
Adequate number of goblet cells with preserved morphology was also
shown. Presence of occasional intraepithelial and subepithelial
eosinophils in tarsal conjunctiva were found in both NP treated and
control eyes, suggesting that the phenomenon is not directly caused
by the administration of the NP formulation.
[0298] The eyes were enucleated and collected immediately after
euthanasia for histpathological evaluation. The entire upper and
lower eyelids were also dissected and collected for evaluation of
the tarsal conjunctiva and underlying soft tissues. Consecutive
sections of the entire ocular globe and eyelids were processed for
microscopic analysis: after initial fixation in 10% neutral
buffered formalin, the tissue was embedded in paraffin, serially
sectioned in 5 .mu.m thick sections, and stained with hematoxylin
and eosin (H&E). The histological slides were evaluated using
bright field microscopy (Leica DM1000, ICC50 HD, Leica Microsystems
Inc, Concord, ON).
10.3 Chronic Response Study Using Dex-b-PLA_PBA NPs
[0299] To analyze the long-term biocompatibility of the NP
formulation, the formulations were administered to rabbit eyes once
a week for up to 12 weeks, while having contralateral eyes as
control, and daily slit-lamp examination was performed similar to
above. Similar to acute response study, no sign of conjunctival
swelling, corneal opacification, or infiltrate were observed in any
of the rabbits at any point of the time during the study. Overall,
the difference in values between the NP treated and control eyes
were insignificant across all of the 7 categories throughout the
duration of the study (12 weeks) (FIG. 25).
[0300] Five female rabbits (New Zealand Albino) were used for this
study. The rabbits were acclimated for one week prior to the
experiment. The nanoparticles are prepared using the
nanoprecipitation method described in Example 4 but without the
drug. The nanoparticles were filtered using 200 nm syringe filter,
and further sterilized using UV irradiation inside a BioSafety
Cabinet (BSC) for 1 hour. One eye was administered with NPs (28
.mu.l; 19 .mu.g of Dex-b-PLA_PBA NPs) once a week for 12 weeks
while the contra-lateral eye is used as control. Slit lamp
examination at 0, 1, 24, 48 hr after administration each week was
used to evaluate 7 different categories (Note that 0 hr means
before administration). These 7 categories (discomfort,
conjunctival redness and swelling, lid swelling, discharge, corneal
opacification, and infiltrate) were graded from 0 (no sign) to 4
(severe). After 12 weeks, the rabbits were euthanized, and the
ocular tissues were collected for further histopathology
analysis.
10.4 Chronic Chronic Response Study Using Dex-b-PLA_PBA NPs
Encapsulated with Cyclosporine A
[0301] Similarly, the long-term biocompatibility of the NP
formulation with encapsulation of Cyclosporine A was also examined
using slit-lamp. The formulation containing both the Dex-b-PLA_PBA
NPs and Cyclosporine A were administered to rabbit eyes, while
having contralateral eyes as control. The slit-lamp examination for
up to 4 weeks has shown no significant difference between the NP
treated and the control eyes in any of the 7 categories (FIG.
26).
[0302] Four female rabbits (New Zealand Albino) were used for this
study. The rabbits were acclimated for one week prior to the
experiment. The nanoparticles are prepared using the
nanoprecipitation method described in Example 4 with Cyclosporine
A. The nanoparticles were filtered using 200 nm syringe filter, and
further sterilized using UV irradiation inside a BioSafety Cabinet
(BSC) for 1 hour. One eye was administered with NPs (28 .mu.l; 19
.mu.g of Dex-b-PLA_PBA NPs and 8 .mu.g of Cyclosporine A) once a
week for 12 weeks, while the contra-lateral eye is used as control.
Slit lamp examination at 0, 1, 24, 48 hr after administration each
week was used to evaluate 7 different categories (Note that 0 hr
means before administration). These 7 categories (discomfort,
conjunctival redness and swelling, lid swelling, discharge, corneal
opacification, and infiltrate) were graded from 0 (no sign) to 4
(severe). After 12 weeks, the rabbits were euthanized, and the
ocular tissues were collected for further histopathology
analysis.
Example 11
In Vitro Nanoparticle Partition Across Tear Fluid Lipid Layer
[0303] The ability of different types of NPs to partition across
tear fluid lipid membrane was studied using in vitro model. The
types of surface properties seem to have very little effect on the
% of partition nanoparticle-drug complexes across the lipid
membrane, with all of them achieve partition of approximately 70%.
However, further studies are required to better simulate the
structure and the turnover phenomenon of the lipid layer in the
tear fluid.
[0304] Artificial tear fluid with lipid layer was prepared using
previously reported method by preparing complex salt solution (CSS)
and lipid stock solution (LSS) (Lorentz, 2009). Add LSS into 2000
fold volume of CSS and bath sonicate at 37.degree. C. for 30
minutes. The mixture is allowed to settle overnight to form the
lipid layer. Nanoparticles with Natamycin were prepared using the
nanoprecipitation method described above. 1 ml of the NP-Natamycin
formulation was added onto 2 ml of CSS/LSS mixture. The mixture was
then incubated at 37.degree. C. for 10 minutes. The bottom 1.5 ml
of the mixture was extracted without disturbing the top layer, and
dried overnight in vacuum desiccator. The precipitates were
dissolved in DMSO again and UV-vis absorption was performed to
calculate the concentration of Natamycin.
Discussion
[0305] The present inventors synthesized a model linear block
copolymer using PLA and Dextran (Dex-b-PLA), and demonstrated that
NPs composed of Dex-b-PLA can self-assemble into core-shell
structured NPs of small particle size, e.g. sizes less than 40 nm,
without using any flow-focusing devices. They further showed that
the size of Dex-b-PLA NPs can be precisely fine-tuned, e.g. between
15-70 nm by altering the molecular weight of the component blocks
(Verma, 2012). Dextran, a natural polysaccharide composed of
1.fwdarw.6 linked .alpha.-D-glucopyranosyl units, was selected as a
model hydrophilic block because of its high hydrophilicity and
biocompatibility. Dextran has an abundance of functional hydroxyl
groups on its back bone. The higher density of surface functional
groups (as opposed to PEG, which has one functional group per
chain) can improve the efficiency of surface functionalization, and
thus, desirable surface properties are more easily achieved with
Dextran based NPs. Dextran coated NPs showed excellent colloidal
stability in physiological media in vitro and long retention in the
systemic circulation in vivo (Verma, 2012; Albert, 1990).
[0306] There is a another fundamental difference in the structure
of NPs composed of Dextran-PLA particles and PEG-PLA particles,
which is due to the greater hydrophilicity of the Dextrans compared
to that of PEG. The more hydrophilic Dextran is less likely
randomly associated in the hydrophobic core of the NPs compared to
PEG, which could also explain the increased drug encapsulated in
the Dextran-PLA NPs compared to PLGA-PEG NPs (Verma, 2012).
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[0376] All publications, patents, and patent applications cited
herein are hereby incorporated by reference in their entirety for
all purposes.
[0377] The above-described embodiments are intended to be examples
only. Alterations, modifications and variations can be effected to
the particular embodiments by those of skill in the art without
departing from the scope of the disclosure, which is defined solely
by the claims appended hereto.
* * * * *