U.S. patent application number 13/465793 was filed with the patent office on 2012-10-11 for nanoparticles for protein drug delivery.
Invention is credited to Zi-Xian Liao, Shu-Fen Peng, Hsing-Wen Sung, Hosheng Tu.
Application Number | 20120258176 13/465793 |
Document ID | / |
Family ID | 46086266 |
Filed Date | 2012-10-11 |
United States Patent
Application |
20120258176 |
Kind Code |
A1 |
Sung; Hsing-Wen ; et
al. |
October 11, 2012 |
NANOPARTICLES FOR PROTEIN DRUG DELIVERY
Abstract
The invention discloses particulate complexes composed of
chitosan, poly-glutamic acid, and at least one bioactive agent,
wherein equal moles of the positively charged chitosan and the
negatively charged poly-glutamic acid substrate form an
electrostatic network enabling improved loading the bioactive
agent.
Inventors: |
Sung; Hsing-Wen; (Hsinchu,
TW) ; Liao; Zi-Xian; (Hsinchu, TW) ; Peng;
Shu-Fen; (Hsinchu, TW) ; Tu; Hosheng; (Newport
Beach, CA) |
Family ID: |
46086266 |
Appl. No.: |
13/465793 |
Filed: |
May 7, 2012 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
13356583 |
Jan 23, 2012 |
8187570 |
|
|
13465793 |
|
|
|
|
13134798 |
Jun 17, 2011 |
8153153 |
|
|
13356583 |
|
|
|
|
12931202 |
Jan 26, 2011 |
7993624 |
|
|
13134798 |
|
|
|
|
12800848 |
May 24, 2010 |
7879313 |
|
|
12931202 |
|
|
|
|
12321855 |
Jan 26, 2009 |
7871988 |
|
|
12800848 |
|
|
|
|
12286504 |
Sep 30, 2008 |
7604795 |
|
|
12321855 |
|
|
|
|
12151230 |
May 5, 2008 |
7541046 |
|
|
12286504 |
|
|
|
|
11398145 |
Apr 5, 2006 |
7381716 |
|
|
12151230 |
|
|
|
|
11284734 |
Nov 21, 2005 |
7282194 |
|
|
11398145 |
|
|
|
|
11029082 |
Jan 4, 2005 |
7265090 |
|
|
11284734 |
|
|
|
|
Current U.S.
Class: |
424/491 ;
424/85.1; 424/85.2; 424/85.4; 424/94.4; 514/1.1; 514/10.1;
514/10.7; 514/10.9; 514/11.3; 514/11.4; 514/11.6; 514/11.9;
514/18.5; 514/20.5; 514/215; 514/297; 514/319; 514/43; 514/44A;
514/44R; 514/490; 514/5.9; 514/52; 514/56; 514/567; 514/662;
514/7.7; 514/9.7; 514/9.9; 977/773; 977/906 |
Current CPC
Class: |
A61P 31/18 20180101;
A61P 25/00 20180101; A61P 29/00 20180101; A61P 25/28 20180101; A61P
39/06 20180101; A61P 27/02 20180101; A61P 25/08 20180101; A61K
31/13 20130101; A61P 31/04 20180101; A61P 3/10 20180101; A61P 31/12
20180101; A61K 31/445 20130101; A61K 31/55 20130101; A61K 9/0043
20130101; A61K 9/5161 20130101; A61P 35/00 20180101; A61K 9/5146
20130101 |
Class at
Publication: |
424/491 ;
514/44.R; 514/44.A; 514/1.1; 514/43; 514/52; 514/56; 514/11.3;
514/11.4; 514/662; 514/319; 514/490; 514/5.9; 514/215; 514/297;
514/11.9; 514/20.5; 514/9.9; 514/10.1; 424/94.4; 424/85.2;
424/85.4; 424/85.1; 514/11.6; 514/567; 514/18.5; 514/9.7; 514/10.9;
514/7.7; 514/10.7; 977/773; 977/906 |
International
Class: |
A61K 9/14 20060101
A61K009/14; A61K 31/713 20060101 A61K031/713; A61K 31/7105 20060101
A61K031/7105; A61K 38/02 20060101 A61K038/02; A61K 31/7052 20060101
A61K031/7052; A61K 31/727 20060101 A61K031/727; A61K 38/27 20060101
A61K038/27; A61K 31/13 20060101 A61K031/13; A61K 31/445 20060101
A61K031/445; A61K 31/27 20060101 A61K031/27; A61K 38/28 20060101
A61K038/28; A61K 31/55 20060101 A61K031/55; A61K 31/473 20060101
A61K031/473; A61K 38/23 20060101 A61K038/23; A61K 38/13 20060101
A61K038/13; A61K 38/24 20060101 A61K038/24; A61K 38/44 20060101
A61K038/44; A61K 38/20 20060101 A61K038/20; A61K 38/21 20060101
A61K038/21; A61K 38/19 20060101 A61K038/19; A61K 48/00 20060101
A61K048/00; A61K 38/11 20060101 A61K038/11; A61K 31/198 20060101
A61K031/198; A61K 38/08 20060101 A61K038/08; A61K 38/06 20060101
A61K038/06; A61K 38/18 20060101 A61K038/18; A61K 38/34 20060101
A61K038/34; A61P 31/12 20060101 A61P031/12; A61P 35/00 20060101
A61P035/00; A61P 31/04 20060101 A61P031/04; A61P 29/00 20060101
A61P029/00; A61P 25/28 20060101 A61P025/28; A61P 25/08 20060101
A61P025/08; A61P 31/18 20060101 A61P031/18; A61P 39/06 20060101
A61P039/06; A61P 27/02 20060101 A61P027/02; A61P 25/00 20060101
A61P025/00; A61P 3/10 20060101 A61P003/10; A61K 31/711 20060101
A61K031/711 |
Claims
1-20. (canceled)
21. A method of delivering at least one bioactive agent to an
animal subject with enhanced cellular uptake of said at least one
bioactive agent by said subject, the method comprising: (a) loading
said bioactive agent in particulate complexes, wherein the
particulate complexes comprise a core portion of positively charged
chitosan, an outer portion of a negatively charged substrate,
optionally a zero-charge compound, and said bioactive agent loaded
in the core portion, wherein said particulate complexes have a mean
complex size between about 50 and 400 nanometers; and (b)
delivering said particulate complexes via a parenteral route to
said animal subject.
22. The method of claim 21, wherein said chitosan is N-trimethyl
chitosan, mono-N-carboxymethyl chitosan (MCC), N-palmitoyl chitosan
(NPCS), EDTA-chitosan, low molecular weight chitosan, chitosan
derivatives, or combinations thereof.
23. The method of claim 21, wherein said at least one bioactive
agent is DNA, RNA, or a small interfering ribonucleic acid
(siRNA).
24. The method of claim 21, wherein said core portion further
comprises micelles.
25. The method of claim 24, wherein the micelles are oil-in-water
micelles, water-in-oil micelles, or hybrid micelles.
26. The method of claim 21, wherein said negatively charged
substrate is selected from the group consisting of .gamma.-PGA,
.alpha.-PGA, PGA-complexone conjugate, water-soluble salts of PGA,
metal salts of PGA, and glycosaminoglycans.
27. The method of claim 21, wherein said particulate complexes are
freeze-dried, thereby said particulate complexes being in a powder
form.
28. The method of claim 21, wherein said particulate complexes are
mixed with trehalose and then freeze-dried, thereby said
particulate complexes being in a powder form.
29. The method of claim 21, wherein said particulate complexes are
treated with an enteric polymer.
30. The method of claim 21, wherein said bioactive agent is
selected from the group consisting of proteins, peptides,
nucleosides, nucleotides, antiviral agents, antineoplastic agents,
antibiotics, and anti-inflammatory drugs.
31. The method of claim 21, wherein said bioactive agent is an
agent for treating Alzheimer's disease selected from the group
consisting of memantine hydrochloride, donepezil hydrochloride,
rivastigmine tartrate, galantamine hydrochloride, insulin, and
tacrine hydrochloride.
32. The method of claim 21, wherein said bioactive agent is
selected from the group consisting of anti-epileptic drugs,
anti-HIV drugs, anti-oxidants, anti-neuromyelitis optica drugs,
meningitis antagonist, and anti-multiple sclerosis drugs.
33. The method of claim 21, wherein said bioactive agent is an
anti-diabetic compound.
34. The method of claim 21, wherein said one bioactive agent is
heparin or low molecular weight heparin.
35. The method of claim 21, wherein said bioactive agent is
selected from the group consisting of hormone, growth hormone, and
human growth hormone.
36. The method of claim 21, wherein said bioactive agent is
selected from the group consisting of calcitonin, cyclosporin,
insulin, oxytocin, tyrosine, enkephalin, tyrotropin releasing
hormone, follicle stimulating hormone, luteinizing hormone,
vasopressin and vasopressin analogs, catalase, superoxide
dismutase, interleukin-II, interferon, colony stimulating factor,
tumor necrosis factor, anti tumor necrosis factor, erythropoietin,
and melanocyte-stimulating hormone.
37. The method of claim 21, a wherein said particulate complexes
are manufactured via a simple and mild ionic gelation method.
38. The method of claim 21, wherein said cellular uptake is via an
endocytosis method.
39. The method of claim 21, a wherein said bioactive agent is a
gene.
40. The method of claim 21, wherein a surface zeta potential of the
particulate complexes is with a positive, neutral, or negative
value.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation application of U.S.
patent application Ser. No. 13/356,583, filed Jan. 23, 2012, which
is a continuation-in-part application of U.S. patent application
Ser. No. 13/134,798, filed Jun. 17, 2011, now U.S. Pat. No.
8,153,153, which is a continuation-in-part application of U.S.
patent application Ser. No. 12/931,202, filed Jan. 26, 2011, now
U.S. Pat. No. 7,993,624, which is a continuation application of
U.S. patent application Ser. No. 12/800,848, filed May 24, 2010,
now U.S. Pat. No. 7,879,313, which is a continuation-in-part
application of U.S. patent application Ser. No. 12/321,855, filed
Jan. 26, 2009, now U.S. Pat. No. 7,871,988, which is a
continuation-in-part application of U.S. patent application Ser.
No. 12/286,504, filed Sep. 30, 2008, now U.S. Pat. No. 7,604,795,
which is a continuation-in-part application of U.S. patent
application Ser. No. 12/151,230, filed May 5, 2008, now U.S. Pat.
No. 7,541,046, which is a continuation-in-part application of U.S.
patent application Ser. No. 11/398,145, filed Apr. 5, 2006, now
U.S. Pat. No. 7,381,716, which is a continuation-in-part
application of U.S. patent application Ser. No. 11/284,734, filed
Nov. 21, 2005, now U.S. Pat. No. 7,282,194, which is a
continuation-in-part application of U.S. patent application Ser.
No. 11/029,082, filed Jan. 4, 2005, now U.S. Pat. No. 7,265,090,
the entire contents of which are incorporated herein by reference.
This application also claims the benefits of a provisional patent
application Ser. No. 61/269,424, filed Jun. 24, 2009.
FIELD OF THE INVENTION
[0002] The present invention is related to medical uses of
nanoparticles having a pharmaceutical composition of chitosan and
polyglutamic acid with bioactive agents and delivery means of the
pharmaceutical composition with enhanced permeability.
BACKGROUND OF THE INVENTION
[0003] Production of pharmaceutically active peptides and proteins
in large quantities has become feasible (Biomacromolecules 2004;
5:1917-1925). The oral route is considered the most convenient way
of drug administrations for patients. Nevertheless, the intestinal
epithelium is a major barrier to the absorption of hydrophilic
drugs such as peptides and proteins (J. Control. Release 1996;
39:131-138). This is because hydrophilic drugs cannot easily
diffuse across the cells through the lipid-bilayer cell membranes.
Attentions have been given to improving paracellular transport of
hydrophilic drugs (J. Control. Release 1998; 51:35-46). The
transport of hydrophilic molecules via the paracellular pathway is,
however, severely restricted by the presence of tight junctions
that are located at the luminal aspect of adjacent epithelial cells
(Annu. Rev. Nutr. 1995; 15:35-55). These tight junctions form a
barrier that limits the paracellular diffusion of hydrophilic
molecules. The structure and function of tight junctions is
described, inter alia, in Ann. Rev. Physiol. 1998; 60:121-160 and
in Ballard T S et al., Annu. Rev. Nutr. 1995; 15:35-55. Tight
junctions do not form a rigid barrier but play an important role in
the diffusion or translocation through the intestinal epithelium
from lumen to bloodstream and vice versa.
[0004] Movement of solutes between cells, through the tight
junctions that bind cells together into a layer as with the
epithelial cells of the gastrointestinal tract, is termed
paracellular transport. Paracellular transport is passive.
Paracellular transport depends on electrochemical gradients
generated by transcellular transport and on solvent drag through
tight junctions. Tight junctions form an intercellular barrier that
separates the apical and basolateral fluid compartments of a cell
layer. Movement of a solute through a tight junction from apical to
basolateral compartments depends on the "tightness" of the tight
junction for that solute.
[0005] Polymeric nanoparticles have been widely investigated as
carriers for drug delivery (Biomaterials 2002; 23:3193-3201). Much
attention has been given to the nanoparticles made of synthetic
biodegradable polymers such as poly-.epsilon.-caprolactone and
polylactide due to their good biocompatibility (J. Drug Delivery
2000; 7:215-232; Eur. J. Pharm. Biopharm. 1995; 41:19-25). However,
these nanoparticles are not ideal carriers for hydrophilic drugs
because of their hydrophobic property. Some aspects of the
invention relate to a novel nanoparticle system, composed of
hydrophilic chitosan and poly(glutamic acid) hydrogels that is
prepared by a simple ionic-gelation method. This technique is
promising as the nanoparticles are prepared under mild conditions
without using harmful solvents. It is known that organic solvents
may cause degradation of peptide or protein drugs that are unstable
and sensitive to their environments (J. Control. Release 2001;
73:279-291).
[0006] Following the oral drug delivery route, protein drugs are
readily degraded by the low pH of gastric medium in the stomach.
The absorption of protein drugs following oral administration is
challenging due to their high molecular weight, hydrophilicity, and
susceptibility to enzymatic inactivation. Protein drugs at the
intestinal epithelium could not partition into the hydrophobic
membrane and thus can only traverse the epithelial barrier via the
paracellular pathway. However, the tight junction forms a barrier
that limits the paracellular diffusion of hydrophilic
molecules.
[0007] Chitosan (CS), a cationic polysaccharide, is generally
derived from chitin by alkaline deacetylation (J. Control. Release
2004; 96:285-300). It was reported from literature that CS is
non-toxic and soft-tissue compatible (Biomacromolecules 2004;
5:1917-1925; Biomacromolecules 2004; 5:828-833). Additionally, it
is known that CS has a special feature of adhering to the mucosal
surface and transiently opening the tight junctions between
epithelial cells (Pharm. Res. 1994; 11:1358-1361). Most
commercially available CSs have a quite large molecular weight (MW)
and need to be dissolved in an acetic acid solution at a pH value
of approximately 4.0 or lower that is sometimes impractical.
However, there are potential applications of CS in which a low MW
would be essential. Given a low MW, the polycationic characteristic
of CS can be used together with a good solubility at a pH value
close to physiological ranges (Eur. J. Pharm. Biopharm. 2004;
57:101-105). Loading of peptide or protein drugs at physiological
pH ranges would preserve their bioactivity. On this basis, a low-MW
CS, obtained by depolymerizing a commercially available CS using
cellulase, is disclosed herein to prepare nanoparticles of the
present invention.
[0008] The .gamma.-PGA, an anionic peptide, is a natural compound
produced as capsular substance or as slime by members of the genus
Bacillus (Crit. Rev. Biotechnol. 2001; 21:219-232). .gamma.-PGA is
unique in that it is composed of naturally occurring L-glutamic
acid linked together through amide bonds. It was reported from
literature that this naturally occurring .gamma.-PGA is a
water-soluble, biodegradable, and non-toxic polymer. A related, but
structurally different polymer, [poly(.alpha.-glutamic acid),
.alpha.-PGA] has been used for drug delivery (Adv. Drug Deliver.
Rev. 2002; 54:695-713; Cancer Res. 1998; 58:2404-2409). .alpha.-PGA
is usually synthesized from poly(.gamma.-benzyl-L-glutamate) by
removing the benzyl protecting group with the use of hydrogen
bromide. Hashida et al. used .alpha.-PGA as a polymeric backbone
and galactose moiety as a ligand to target hepatocytes (J. Control.
Release 1999; 62:253-262). Their in vivo results indicated that the
galactosylated .alpha.-PGA had a remarkable targeting ability to
hepatocytes and degradation of .alpha.-PGA was observed in the
liver.
[0009] Thanou et al. reported chitosan and its derivatives as
intestinal absorption enhancers (Adv Drug Deliv Rev 2001;
50:S91-S101). Chitosan, when protonated at an acidic pH, is able to
increase the paracellular permeability of peptide drugs across
mucosal epithelia. Co-administration of chitosan or trimethyl
chitosan chloride with peptide drugs were found to substantially
increase the bioavailability of the peptide in animals compared
with administrations without the chitosan component.
[0010] Fernandez-Urrusuno et al. reported that chitosan
nanoparticles enhanced the nasal absorption of insulin to a greater
extent than an aqueous solution of chitosan (Pharmaceutical
Research 1999; 16:1576-1581), entire contents of which are
incorporated herein by reference. Insulin-loaded chitosan
nanoparticles displayed a high positive charge and a rapid insulin
release kinetics properties, which render them very interesting
systems for nasal drug delivery.
[0011] Heppe et al. in U.S. patent application publication no.
2006/0051423 A1, entire contents of which are incorporated herein
by reference, discloses a chitosan-based transport system for
overcoming the blood-brain barrier. This transport system can
convey active agents or markers into the brain. The transport
system contains at least one substance selected from the group
consisting of chitin, chitosan, chitosan oligosaccharides,
glucosamine, and derivatives thereof, and optionally one or more
active agents and/or one or more markers and/or one or more
ligands. However, Heppe et al. neither teaches a chitosan-shelled
nanoparticle transport system, nor asserts substantial efficacy of
chitosan-shelled nanoparticles permeating through blood-brain
barriers.
[0012] van der Lubben et al. reported that chitosan and its
derivatives are effective and safe absorption enhancers to improve
mucosal (nasal, peroral) delivery of hydrophilic macromolecules
such as protein and peptide drugs and vaccines (Euro J Pharma Sci
2001; 14:201-207), entire contents of which are incorporated herein
by reference. Interaction of the positively charged amino group at
the C-2 position of chitosan with the negatively charged sites on
the cell surface and tight junctions allows paracellular transport
of large hydrophilic compounds by opening the tight junctions of
the membrane barrier.
[0013] Minn et al. reported drug transport into the mammalian brain
via the nasal pathway (J Drug Targeting 2002; 10:285-296), entire
contents of which are incorporated herein by reference. The rate of
entry into and distribution of drugs and other xenobiotics within
the central nervous system depends on the particular anatomy of the
brain microvessels forming the blood-brain barrier and of the
choroids plexus forming the blood-cerebrospinal fluid barrier,
which possess tight junctions preventing the passage of most polar
substances.
[0014] Vyas et al. reported a preliminary study on brain targeting
for intranasal mucoadhesive microemulsions of clonazepam (J Pharma
Sci 2006; 95:570-580) entire contents of which are incorporated
herein by reference. In the rabbit study, it shows more effective
brain targeting with intranasal administration than intravenous
administration. Rabbit brain scintigraphy also showed higher
intranasal uptake of the drug into the brain.
[0015] Prokop et al in U.S. Pat. No. 6,383,478 teaches
nanoparticles in the range of 1-1000 nm diameter for drug delivery
comprising at least two or more polyanions, one or more
polycation(s), one or more small cation(s), and the drug. It was
disclosed that at least two polyanions (for example, alginate plus
other polyanions) are required in a polymeric drug delivery vehicle
to deliver protein factors.
[0016] However, none of the above prior art teach a pharmaceutical
composition of novel nanoparticles in a size less than 400
nanometers for an animal subject, the nanoparticles comprising
positively charged chitosan and a negatively charged substrate
through the nanoparticle structure, wherein the negatively charged
substrate is substantially neutralized with the positively charged
chitosan to enhance loading of at least one bioactive agent that is
loaded within the nanoparticles.
SUMMARY OF THE INVENTION
[0017] It is one object of the present invention to provide a novel
nanoparticle system and methods of preparation for paracellular
transport drug delivery using a simple and mild ionic-gelation
method upon addition of, for example, a poly-.gamma.-glutamic acid
(.gamma.-PGA) solution, into regular molecular weight chitosan
solution. In one embodiment, the chitosan employed is N-trimethyl
chitosan (TMC), EDTA-chitosan, mono-N-carboxymethyl chitosan (MCC),
N-palmitoyl chitosan (NPCS), chitosan derivatives, or combinations
thereof. In an alternate embodiment, the chitosan employed is low
molecular weight chitosan (low-MW CS). In one embodiment, the
molecular weight of a low-MW CS of the present invention is about
80 kDa or less, preferably at about 40 kDa, adapted for adequate
solubility at a pH that maintains the bioactivity of protein and
peptide drugs. It is stipulated that a chitosan particle with about
30-50 kDa molecular weight is kidney inert. The particle size and
the zeta potential value of the prepared nanoparticles are
controlled by their constituted compositions. The results obtained
by the TEM (transmission electron microscopy) and AFM (atomic force
microscopy) examinations showed that the morphology of the prepared
nanoparticles was generally spherical or spheroidal in shape.
[0018] Evaluation of the prepared nanoparticles in enhancing
intestinal paracellular transport was investigated in vitro in
Caco-2 cell monolayers model. Some aspects of the present invention
provide the nanoparticles with CS dominated on the surfaces to
effectively reduce the transepithelial electrical resistance (TEER)
of Caco-2 cell monolayers. The confocal laser scanning microscopy
(CLSM) observations confirm that the nanoparticles or fragments
thereof with CS dominating on the surface are able to open the
tight junctions between Caco-2 cells and allows transport of the
nanoparticles via the paracellular pathways.
[0019] Some aspects of the invention relate to a method of
enhancing intestinal or blood brain paracellular transport
configured for delivering at least one bioactive agent or API
(active pharmaceutical ingredient) in a patient comprising
administering nanoparticles composed of .gamma.-PGA and chitosan,
wherein the step of administering the nanoparticles may be via oral
administration (including sublingual, buccal, cheek and the like),
nasal instillation, transcutaneous injection, or injection into a
blood vessel.
[0020] In one embodiment, the surface of the nanoparticles
comprises about equal quantity or moles of chitosan and a
negatively charged substrate so the surface potential is about
neutral or zero. In another embodiment, a substantial surface of
the nanoparticles is characterized with a positive surface charge
or negative surface charge. In one embodiment, substantially all of
the negatively charged substrate conjugates with substantially all
of the positively charged chitosan so to maintain a substantially
zero-charge (neutral) nanoparticle. The conjugation of the
nanoparticle components enhances API loading content for the
current nanoparticle system. In one embodiment, at least one
bioactive, a protein drug, or a third compound is loaded within the
nanoparticle system.
[0021] In a further embodiment, the nanoparticles have a mean
particle size between about 50 and 400 nanometers, preferably
between about 100 and 300 nanometers, and most preferably between
about 100 and 200 nanometers. The bioactive nanoparticle fragments
resulting from the nanoparticles of the present invention are
generally in the range of about 10 to 150 nm, preferably in the
range of about 20 to 100 nm, and most preferably in the range of
about 20 to 50 nm.
[0022] In some embodiments, the nanoparticles are loaded with a
therapeutically effective amount of at least one bioactive agent,
wherein the bioactive agent is selected from the group consisting
of proteins, peptides, nucleosides, nucleotides, antiviral agents,
antineoplastic agents, antibiotics, and anti-inflammatory drugs. In
another embodiment, the bioactive agent is associated with or
entrapped within micelles before being loaded into the nanoparticle
structure. In still another embodiment, the bioactive agent is
lipophilic, hydrophobic, or hydrophilic. After being associated
with micelles (via a physical, chemical, or a biochemical means),
the lipophilic hydrophobic or hydrophilic bioactive agent becomes
encapsulatable through the affinity of micelles toward the inactive
ingredients of chitosan and a negative charged substrate of the
nanoparticles. In one embodiment, the micelles are either
oil-in-water micelles or water-in-oil micelles.
[0023] Further, the bioactive agent may be selected from the group
consisting of calcitonin, cyclosporin, insulin, oxytocin, tyrosine,
enkephalin, tyrotropin releasing hormone, follicle stimulating
hormone, luteinizing hormone, vasopressin and vasopressin analogs,
catalase, superoxide dismutase, interleukin-II, interferon, colony
stimulating factor, tumor necrosis factor, anti tumor necrosis
factor, erythropoietin, and melanocyte-stimulating hormone. In one
preferred embodiment, the bioactive agent is an Alzheimer
antagonist for treating Alzheimer's diseases.
[0024] Some aspects of the invention relate to a dose of
nanoparticles that effectively enhance intestinal or blood brain
paracellular transport comprising a negatively charged component,
such as .gamma.-PGA, .alpha.-PGA, PGA-DTPA, heparin, or heparan
sulfate, in the core and low molecular weight chitosan.
[0025] In a further embodiment, the nanoparticles comprise at least
one bioactive agent, such as anti-diabetic compound (for example,
insulin, insulin analog, GLP-1, GLP-1 analog, and the like),
Alzheimer's disease antagonist, Parkison's disease antagonist, or
other protein/peptide. The bioactive agent for treating Alzheimer's
disease may include memantine hydrochloride (Axura.RTM. by Merz
Pharmaceuticals), donepezil hydrochloride (Aricept.RTM. by Eisai
Co. Ltd.), rivastigmine tartrate (Exelon.RTM. by Novartis),
galantamine hydrochloride (Reminyl.RTM. by Johnson & Johnson),
and tacrine hydrochloride (Cognex.RTM. by Parke Davis). Examples of
insulin or insulin analog products include, but not limited to,
Humulin.RTM. (by Eli Lilly), Humalog.RTM. (by Eli Lilly) and
Lantus.RTM. (by Aventis).
[0026] Some aspects of the invention relate to a dose of
nanoparticles that effectively enhance intestinal or blood brain
paracellular transport comprising a negatively charged substrate
and low molecular weight chitosan, wherein the nanoparticles are
crosslinked with a crosslinking agent or with light, such as
ultraviolet irradiation.
[0027] Some aspects of the invention provide a dose of
nanoparticles characterized by enhancing intestinal or blood brain
paracellular transport, each nanoparticle comprising a first
component of at least one bioactive agent, a second component of
low molecular weight chitosan, and a third component that is
negatively charged. In one embodiment, the third component is
.gamma.-PGA, .alpha.-PGA, derivatives or salts of PGA, PGA-DTPA,
PGA-complexone conjugate, heparin or alginate. In another
embodiment, the first component comprises insulin at a
concentration range of 0.075 to 0.091 mg/ml, the second component
at a concentration range of 0.67 to 0.83 mg/ml, and the third
component comprises .gamma.-PGA at a concentration range of 0.150
to 0.184 mg/ml.
[0028] Some aspects of the invention provide a dose of
nanoparticles characterized by enhancing intestinal or blood brain
paracellular transport, each nanoparticle comprising a first
component of at least one bioactive agent, a second component of
low molecular weight chitosan, and a third component that is
negatively charged, wherein the at least one bioactive agent is an
antagonist for Alzheimer's disease or is for treating Alzheimer's
disease selected from the group consisting of memantine
hydrochloride, donepezil hydrochloride, rivastigmine tartrate,
galantamine hydrochloride, insulin, and tacrine hydrochloride. In a
further embodiment, the at least one bioactive agent is insulin or
insulin analog. In still another embodiment, the at least one
bioactive agent is selected from the group consisting of proteins,
peptides, nucleosides, nucleotides, antiviral agents,
antineoplastic agents, antibiotics, and anti-inflammatory
drugs.
[0029] Some aspects of the invention provide a dose of
nanoparticles characterized by enhancing intestinal or blood brain
paracellular transport, wherein the nanoparticles are further
encapsulated in a capsule for oral administration. In one
embodiment, the nanoparticles are freeze-dried. In one embodiment,
the interior surface of the capsule is treated to be lipophilic or
hydrophobic. In another embodiment, the exterior surface of the
capsules or tablets is enteric-coated.
[0030] Some aspects of the invention provide a dose of
nanoparticles characterized by enhancing intestinal or blood brain
paracellular transport, each nanoparticle comprising a first
component of at least one bioactive agent, a second component of
low molecular weight chitosan, and a third component that is
negatively charged, wherein the second component is crosslinked. In
one embodiment, the degree of crosslinking is less than 50%. In
another embodiment, the degree of crosslinking is ranged between 1%
and 20%.
[0031] Some aspects of the invention provide a dose of
nanoparticles characterized by enhancing intestinal or blood brain
paracellular transport, each nanoparticle comprising a first
component of at least one bioactive agent, a second component of
low molecular weight chitosan, and a third component that is
negatively charged, wherein the second component is crosslinked
with a crosslinking agent selected from the group consisting of
genipin, its derivatives, analog, stereoisomers and mixtures
thereof. In one embodiment, the crosslinking agent is selected from
the group consisting of epoxy compounds, dialdehyde starch,
glutaraldehyde, formaldehyde, dimethyl suberimidate, carbodiimides,
succinimidyls, diisocyanates, acyl azide, reuterin, ultraviolet
irradiation, dehydrothermal treatment,
tris(hydroxymethyl)phosphine, ascorbate-copper, glucose-lysine and
photo-oxidizers.
[0032] Some aspects of the invention provide a dose or a
pharmaceutical composition of nanoparticles characterized by
enhancing intestinal or blood brain paracellular transport, wherein
the low molecule weight chitosan has a molecular weight of 80 kDa
or less. In one embodiment, the low molecule weight chitosan is
further grafted with a polymer.
[0033] Some aspects of the invention provide a method of enhancing
intestinal or blood brain paracellular transport comprising
administering a dose of nanoparticles, wherein each nanoparticle
comprises a first component of at least one bioactive agent, a
second component of low molecular weight chitosan, and a third
component that is negatively charged. In one embodiment, the step
of administering the dose of nanoparticles is via oral
administration for enhancing intestinal paracellular transport. In
another embodiment, the step of administering the dose of
nanoparticles is via venous administration or injection to a blood
vessel for enhancing blood brain paracellular transport or
mitigating the blood-brain barrier (BBB). In a further embodiment,
the step of administrating the dose of nanoparticles is via nasal
instillation, buccal or oral administration (including sublingual).
In a further embodiment, the step of administrating the dose of
nanoparticles is via endocytosis. The nanoparticles of fragments
thereof may be in a freeze-dried powder form.
[0034] Some aspects of the invention provide a method of treating
diabetes of a patient comprising orally administering anti-diabetic
compound (for example, insulin, insulin analog, GLP-1, GLP-1
analog, anti-diabetic drugs, and the like) containing nanoparticles
with a dosage effective amount of the anti-diabetic compound to
treat the diabetes, wherein at least a portion of the nanoparticles
comprises positively charged chitosan and a negatively charged
substrate in about the equal amount or moles. In another
embodiment, the negatively charged substrate is selected from the
group consisting of .gamma.-PGA, .alpha.-PGA, water-soluble salts
of PGA, metal salts of PGA, PGA-complexone conjugate, heparin,
heparin analogs, low molecular weight heparin, glycosaminoglycans,
and alginate. The molecular formula of the insulin is selected from
the group consisting of C.sub.254H.sub.377N.sub.65O.sub.75S.sub.6,
C.sub.257H.sub.383N.sub.65O.sub.77S.sub.6,
C.sub.256H.sub.381N.sub.65O.sub.79S.sub.6,
C.sub.267H.sub.404N.sub.72O.sub.78S.sub.6,
C.sub.267H.sub.408N.sub.72O.sub.77S.sub.6 (insulin glargine),
C.sub.267H.sub.402N.sub.64O.sub.76S.sub.6 (insulin determir), and
the like.
[0035] In one embodiment, the orally administering insulin
containing nanoparticles comprise a dosage effective amount of the
insulin to treat the diabetes comprising an insulin amount of
between about 15 international units to 45 units, preferably
between about 25 units to 35 units, per kilogram body weight of the
patient. In a further embodiment, the insulin-containing
nanoparticle comprises a trace amount of zinc or calcium, or is
treated with enteric coating. Further, the nanoparticle or
particulate complexes are treated with enteric polymer.
[0036] In one embodiment, the insulin containing nanoparticles
further comprise at least one absorption or permeation enhancer,
wherein the enhancer may be selected from the group consisting of
chelators, EDTA (ethylenediaminetetraacetic acid), bile salts,
surfactants, medium-chain fatty acids, phosphate esters, and the
like. In another embodiment, the nanoparticles and the enhancer are
co-encapsulated in a capsule or are administrated separately.
[0037] Some aspects of the invention provide nanoparticles for oral
administration in a patient, comprising positively charged
chitosan, a negatively charged substrate, and a bioactive agent
conjugated with the substrate, wherein the substrate is selected
from the group consisting of heparin, heparin analogs, low
molecular weight heparin, glycosaminoglycans, and alginate, the
bioactive agent being selected from the group consisting of
chondroitin sulfate, hyaluronic acid, calcitonin, vancomycin,
growth factor and protein with pharmaceutically effective
amount.
[0038] Some aspects of the invention provide a method of treating
Alzheimer's diseases of a patient comprising intravenously
administering or intramuscularly/subcutaneously injecting bioactive
nanoparticles with a dosage effective to treat the Alzheimer's
diseases, wherein the bioactive nanoparticles comprises positively
charged chitosan, a negatively charged substrate, and at least one
bioactive agent for treating Alzheimer's disease, wherein the at
least one bioactive agent is selected from the group consisting of
memantine hydrochloride, donepezil hydrochloride, rivastigmine
tartrate, galantamine hydrochloride, and tacrine hydrochloride.
[0039] In one embodiment, the dosage effective to treat the
Alzheimer's diseases comprises administering the at least one
bioactive agent for treating Alzheimer's disease at about 10 mg to
40 mg per day over a period of one month to one year. In another
embodiment, at least a portion the nanoparticles is crosslinked,
preferably at a degree of crosslinking less than about 50%, or most
preferably between about 1% and 20%.
[0040] One aspect of the invention provides a pharmaceutical
composition of nanoparticles, wherein the nanoparticles may be
freeze-dried to form solid dried nanoparticles. The dried
nanoparticles may be loaded in a capsule or a tablet/pill for oral
administration in a patient, wherein the capsule or tablet/pill may
be further enterically coated. The freeze-dried nanoparticles can
be rehydrated in solution or by contacting fluid so to revert to
wet nanoparticles. In one embodiment, nanoparticles may be mixed
with trehalose or with hexan-1,2,3,4,5,6-hexyl in a freeze-drying
process. In one embodiment, the interior surface of the capsule is
treated to be lipophilic or hydrophobic. In another embodiment, the
exterior surface of the capsule, tablet or pill is
enteric-coated.
[0041] Some aspects of the invention provide a pharmaceutical
composition of nanoparticles characterized by enhancing
paracellular transport, each nanoparticle comprising a shell
component and a core component, wherein at least a portion of the
shell component comprises chitosan and wherein the core component
is comprised of MgSO.sub.4, sodium tripolyphosphate, at least one
bioactive agent, and a negatively charged compound, wherein a
substantial portion of the negatively charged compound is
conjugated to the chitosan. In one embodiment, the negatively
charged component of the pharmaceutical composition is .gamma.-PGA
or a derivative or salt of PGAs.
[0042] Some aspects of the invention provide an orally deliverable
capsule to an animal subject comprising: (a) an empty capsule; and
(b) bioactive nanoparticles loaded within the empty capsule,
wherein the nanoparticles comprise chitosan, a negatively charged
substrate, and at least one bioactive agent. In one embodiment, the
empty capsule comprises a two-part hard gelatin capsule. In another
embodiment, the capsule is treated with an enteric coating.
[0043] One object of the present invention is to provide a method
of manufacturing the orally deliverable capsule, the method
comprising steps of: (a) providing an empty capsule; (b) providing
bioactive nanoparticles, wherein the nanoparticles comprise
chitosan, a negatively charged substrate, and at least one
bioactive agent; (c) freeze-drying the nanoparticles; and (d)
filling the freeze-dried bioactive nanoparticles into the empty
capsule, thereby producing an orally deliverable capsule. In one
embodiment, the bioactive nanoparticles further comprise magnesium
sulfate and TPP.
[0044] Some aspects of the invention provide a pharmaceutical
composition of nanoparticles for oral administration in a patient,
the nanoparticles comprising positively charged chitosan, a
negatively charged substrate, wherein the negatively charged
substrate is at least partially or substantially totally
neutralized with the positively charged chitosan, and at least one
bioactive agent loaded within the nanoparticles. In one embodiment,
the bioactive agent is a non-insulin exenatide, a non-insulin
pramlintide, insulin, insulin analog, or combinations thereof. In
one embodiment, the nanoparticles are formed via a simple and mild
ionic-gelation method.
[0045] In one embodiment of the pharmaceutical composition of the
present invention, the substrate is PGA, wherein the PGA may be
.gamma.-PGA, .alpha.-PGA, PGA derivatives, PGA-DTPA, PGA-complexone
conjugates, or salts of PGA. In one embodiment of the
pharmaceutical composition of the present invention, the substrate
is heparin, wherein the heparin is a low molecular weight
heparin.
[0046] In one embodiment, a surface of the nanoparticles of the
pharmaceutical composition of the present invention is
characterized with a positive surface charge, wherein the
nanoparticles have a surface charge from about +15 mV to about +50
mV. In another embodiment, the nanoparticles have a mean particle
size between about 50 and 400 nanometers. In still another
embodiment, at least a portion of the shell portion of the
nanoparticles is crosslinked. In a further embodiment, the
nanoparticles are in a form of freeze-dried powder. In one
embodiment, the nanoparticles of the pharmaceutical composition of
the present invention further comprise magnesium sulfate and
TPP.
[0047] Some aspects of the invention provide a method of delivering
a bioactive agent to blood circulation in a patient, comprising:
(a) providing nanoparticles according to the pharmaceutical
composition of the present invention, wherein the nanoparticles are
formed via a simple and mild ionic-gelation method; (b)
administering the nanoparticles orally toward the intestine of the
patient; (c) urging the nanoparticles to be absorbed onto a surface
of an epithelial membrane of the intestine; (d) permeating
bioactive agent to pass through an epithelial barrier of the
intestine; and (e) releasing the bioactive agent into the blood
circulation. In one embodiment, the bioactive agent is selected
from the group consisting of exenatide, pramlintide, insulin,
insulin analog, and combinations thereof.
[0048] Some aspects of the invention provide a nanoparticle
delivery system for enhancing the paracellular permeation of at
least one bioactive agent, comprising nanoparticles or fragments
thereof, the nanoparticles comprising a shell portion that is
dominated by positively charged chitosan, a core portion that
contains negatively charged substrate, wherein the negatively
charged substrate is at least partially reacted with a portion of
the positively charged chitosan in the core portion, and the at
least one bioactive agent loaded within the nanoparticles, wherein
the substrate is PGA or heparin.
[0049] In one embodiment, the PGA of the nanoparticle delivery
system is .gamma.-PGA, .alpha.-PGA, derivatives of PGA or salts of
PGA. In another embodiment, a surface of the nanoparticles of the
nanoparticle delivery system is characterized with a positive
surface charge or zero surface charge. In a further embodiment, the
nanoparticles of the nanoparticle delivery system are formed via a
simple and mild ionic-gelation method.
[0050] In one embodiment, the bioactive agent is insulin or insulin
analog. In another embodiment, the bioactive agent is selected from
the group consisting of anti-inflammatory drugs, anti-epileptic
drugs, Alzheimer's antagonist, anti-HIV drugs, anti-oxidants,
anti-neuromyelitis optica drugs, meningitis antagonist, and
anti-multiple sclerosis drugs.
[0051] Some aspects of the invention provide a method for treating
disorders of a tight junction comprising delivering a nanoparticle
delivery system to the tight junction, wherein the nanoparticle
delivery system comprises nanoparticles or fragments thereof
according to a pharmaceutical composition disclosed. In one
embodiment, the bioactive agent is selected from the group
consisting of anti-epileptic drugs, anti-inflammatory drugs,
meningitis antagonist, and anti-oxidant. Some aspects of the
invention provide a method of delivering a bioactive agent through
a carrier of bioactive nanoparticles of fragments thereof to a
brain via intranasal, buccal, sublingual, intravenous, or blood
vessel route.
[0052] Some aspects of the invention provide a pharmaceutical
composition of nanoparticles, the nanoparticles comprising
positively charged chitosan, a negatively charged substrate,
wherein the negatively charged substrate is at least partially or
substantially totally neutralized with the positively charged
chitosan, and micelles, wherein at least one bioactive agent is
loaded within the micelles. In one embodiment, the substrate is
PGA-complexone conjugate. In another embodiment, the micelles are
made via an emulsion process, an oil-in-water microemulsion process
or self-emulsifying process. In still another embodiment, the at
least one bioactive agent is a lipophilic or hydrophobic bioactive
agent.
[0053] Some aspects of the invention provide a pharmaceutical
composition of nanoparticles, the nanoparticles consisting of
positively charged chitosan, a negatively charged substrate,
optionally a zero-charge compound, and at least one active
pharmaceutical ingredient (API) or bioactive agent, wherein the
nanoparticles have a mean particle size between about 50 and 400
nanometers. In one embodiment, the chitosan is N-trimethyl
chitosan, mono-N-carboxymethyl chitosan (MCC), N-palmitoyl chitosan
(NPCS), EDTA-chitosan, low molecular weight chitosan, chitosan
derivatives, or combinations thereof. In another embodiment, the
negatively charged substrate is selected from the group consisting
of .gamma.-PGA, .alpha.-PGA, water-soluble salts of PGA, metal
salts of PGA, glycosaminoglycans, and a PGA-complexone
conjugate.
[0054] Some aspects of the invention provide a pharmaceutical
composition of nanoparticles consisting of positively charged
chitosan, a negatively charged substrate, optionally a zero-charge
compound, and at least an active pharmaceutical agent (API) or
bioactive agent, wherein the nanoparticles are formulated into a
tablet or pill configuration and wherein the tablet or pill is
treated with an enteric coating. In one embodiment, the
nanoparticles are encapsulated in a capsule, wherein optionally the
capsule further comprises a pharmaceutically acceptable carrier,
diluent, excipient, a solubilizer, bubbling agent, or emulsifier.
In another embodiment, the capsule is treated with an enteric
coating, or the capsule further comprises at least one permeation
or absorption enhancer. In a further embodiment, the nanoparticles
are freeze-dried, thereby the nanoparticles being in a powder form.
In one embodiment, the zero-charge compound in the nanoparticles is
a permeation or absorption enhancer.
[0055] Some aspects of the invention provide a pharmaceutical
composition of nanoparticles consisting of positively charged
chitosan, a negatively charged substrate, optionally a zero-charge
compound, and at least an active pharmaceutical agent (API) or
bioactive agent, wherein the at least one API is an anti-diabetic
compound, heparin or low molecular weight heparin, or an
anti-hemophilic factor.
[0056] Some aspects of the invention provide a pharmaceutical
composition of nanoparticles consisting of positively charged
chitosan, a negatively charged substrate, optionally a zero-charge
compound, and at least an active pharmaceutical agent (API) or
bioactive agent, wherein the negatively charged substrate is
heparin or low molecular weight heparin.
BRIEF DESCRIPTION OF THE DRAWINGS
[0057] Additional objects and features of the present invention
will become more apparent and the disclosure itself will be best
understood from the following Detailed Description of the Exemplary
Embodiments, when read with reference to the accompanying
drawings.
[0058] FIG. 1 shows GPC chromatograms of (a) standard-MW CS before
depolymerization and the low-MW CS after depolymerization; and (b)
the purified .gamma.-PGA obtained from microbial fermentation.
[0059] FIG. 2 shows (a) FT-IR and (b) .sup.1H-NMR spectra of the
purified .gamma.-PGA obtained from microbial fermentation.
[0060] FIG. 3 shows FT-IR spectra of the low-MW CS and the prepared
CS-.gamma.-PGA nanoparticles.
[0061] FIG. 4 shows (a) a TEM micrograph of the prepared
CS-.gamma.-PGA nanoparticles (0.10% .gamma.-PGA:0.20% CS) and (b)
an AFM micrograph of the prepared CS-.gamma.-PGA nanoparticles
(0.01% .gamma.-PGA:0.01% CS).
[0062] FIG. 5 shows changes in particle size and zeta potential of
(a) the CS-.gamma.-PGA nanoparticles (0.10% .gamma.-PGA:0.20% CS)
and (b) the CS-.gamma.-PGA nanoparticles (0.10% .gamma.-PGA:0.01%
CS) during storage for up to 6 weeks.
[0063] FIG. 6 shows effects of the prepared CS-.gamma.-PGA
nanoparticles on the TEER values of Caco-2 cell monolayers.
[0064] FIG. 7 shows fluorescence images (taken by an inversed
confocal laser scanning microscope) of 4 optical sections of a
Caco-2 cell monolayer that had been incubated with the
fCS-.gamma.-PGA nanoparticles with a positive surface charge (0.10%
.gamma.-PGA:0.20% CS) for (a) 20 min and (b) 60 min.
[0065] FIG. 8 shows an illustrative protein transport mechanism
through a cell layer, including transcellular transport and
paracelluler transport.
[0066] FIG. 9A-C show a schematic illustration of a paracellular
transport mechanism.
[0067] FIG. 10 shows insulin-loaded nanoparticles with a core
composition consisted of .gamma.-PGA, MgSO.sub.4, sodium
tripolyphosphate (TPP), and insulin.
[0068] FIG. 11 shows loading capacity and association efficiency of
insulin in nanoparticles of chitosan and .gamma.-PGA.
[0069] FIG. 12 shows loading capacity and association efficiency of
insulin in nanoparticles of chitosan as reference.
[0070] FIG. 13 shows the stability of insulin-loaded
nanoparticles.
[0071] FIG. 14 shows a representative in vitro study with insulin
drug release profile in a pH-adjusted solution.
[0072] FIG. 15 shows the effect of insulin of orally administered
insulin-loaded nanoparticles on hypoglycemia in diabetic rats.
[0073] FIG. 16A-C show a proposed mechanism of nanoparticles
released from the enteric coated capsules.
[0074] FIG. 17 shows scheme of a micelle formed by phospholipids in
an aqueous solution.
[0075] FIG. 18 shows scheme of a micelle formed by phospholipids in
an organic solvent.
[0076] FIG. 19 shows the effect of orally administered
insulin-loaded nanoparticles on `glucose reduction %` in diabetic
rats, wherein the freeze-dried nanoparticles were loaded in an
enterically coated capsule upon delivery.
[0077] FIG. 20 shows cross-section of the different structures that
phospholipids can take in an aqueous solution. The circles are the
hydrophilic heads and the wavy lines are the fatty acyl side
chains.
[0078] FIG. 21 shows an in vivo subcutaneous study using insulin
injectables and insulin-containing nanoparticles.
[0079] FIG. 22 shows experimental data on enzyme inhibition study
with (.gamma.-PGA)-DTPA conjugate.
[0080] FIG. 23 shows particle complexes (CS/DNA/.gamma.-PGA) size
and surface zeta potential.
DETAILED DESCRIPTION OF THE EXEMPLARY EMBODIMENTS
[0081] The preferred embodiments of the present invention described
below relate particularly to preparation of nanoparticles composed
of chitosan/poly-glutamic acid/insulin and their permeability to
enhance the intestinal or blood brain paracellular permeation by
opening the tight junctions between epithelial cells. While the
description sets forth various embodiment specific details, it will
be appreciated that the description is illustrative only and should
not be construed in any way as limiting the invention. Furthermore,
various applications of the invention, and modifications thereto,
which may occur to those who are skilled in the art, are also
encompassed by the general concepts described below.
[0082] .gamma.-PGA is a naturally occurring anionic homo-polyamide
that is made of L-glutamic acid units connected by amide linkages
between .alpha.-amino and .gamma.-carboxylic acid groups. It is an
exocellular polymer of certain Bacillus species that is produced
within cells via the TCA cycle and is freely excreted into the
fermentation broth. Its exact biological role is not fully known,
although it is likely that .gamma.-PGA is linked to increasing the
survival of producing strains when exposed to environmental
stresses. Because of its water-solubility, biodegradability,
edibility, and non-toxicity toward humans and the environment,
several applications of .gamma.-PGA in food, cosmetics, medicine,
and water treatment have been investigated in the past few
years.
Example No. 1
Materials and Methods of Nanoparticles Preparation
[0083] CS (MW .about.2.8.times.10.sup.5) with a degree of
deacetylation of approximately 85% was acquired from Challenge
Bioproducts Co. (Taichung, Taiwan). Acetic acid, cellulase (1.92
units/mg), fluorescein isothiocyanate (FITC), phosphate buffered
saline (PBS), periodic acid, sodium acetate, formaldehyde, bismuth
subnitrate, and Hanks balanced salt solution (HESS) were purchased
from Sigma Chemical Co. (St. Louis, Mo.). Ethanol absolute
anhydrous and potassium sodium tartrate were obtained from Merck
(Darmstadt, Germany). Non-essential amino acid (NEAA) solution,
fetal bovine serum (FBS), gentamicin and trypsin-EDTA were acquired
from Gibco (Grand Island, N.Y.). Eagle's minimal essential medium
(MEM) was purchased from Bio West (Nuaille, France). All other
chemicals and reagents used were of analytical grade.
Example No. 2
Depolymerization of CS by Enzymatic Hydrolysis
[0084] Regular CS was treated with enzyme (cellulase) to produce
low-MW CS according to a method described by Qin et al. with some
modifications (Food Chem. 2004; 84:107-115). A solution of CS (20
WI) was prepared by dissolving CS in 2% acetic acid. Care was taken
to ensure total solubility of CS. Then, the CS solution was
introduced into a vessel and adjusted to the desired pH 5.0 with 2N
aqueous NaOH. Subsequently, cellulase (0.1 g) was added into the CS
solution (100 ml) and continuously stirred at 37.degree. C. for 12
hours. Afterward, the depolymerized CS was precipitated with
aqueous NaOH at pH 7.0-7.2 and the precipitated CS was washed three
times with deionized water. The resulting low-MW CS was lyophilized
in a freeze dryer (Eyela Co. Ltd, Tokyo, Japan).
[0085] The average molecular weight of the depolymerized CS was
determined by a gel permeation chromatography (GPC) system equipped
with a series of PL aquagel-OH columns (one Guard 8 .mu.M,
50.times.7.5 mm and two MIXED 8 .mu.m, 300.times.7.5 mm, PL
Laboratories, UK) and a refractive index (RI) detector (RI2000-F,
SFD, Torrance, Calif.). Polysaccharide standards (molecular weights
range from 180 to 788,000, Polymer Laboratories, UK) were used to
construct a calibration curve. The mobile phase contained 0.01M
NaH.sub.2PO.sub.4 and 0.5M NaNO.sub.3 and was brought to a pH of
2.0. The flow rate of mobile phase was 1.0 ml/min, and the columns
and the RI detector cell were maintained at 30.degree. C.
[0086] Factors limiting applications of most commercially available
CSs are their high molecular weight and thus high viscosity and
poor solubility at physiological pH ranges. Low-MW CS overcomes
these limitations and hence finds much wider applications in
diversified fields. It was suggested that low-MW CS be used as a
parenteral drug carrier due to its lower antigen effect (Eur. J.
Pharm. Biopharm. 2004; 57:101-105). Low-MW CS was used as a
non-viral gene delivery system and showed promising results.
[0087] FIG. 1a shows GPC chromatograms of both standard-MW (also
known as regular-MW) and low-MW CS. It is known that cellulase
catalyzes the cleavage of the glycosidic linkage in CS. The low-MW
CS used in the study was obtained by precipitating the
depolymerized CS solution with aqueous NaOH at pH 7.0-7.2. Thus,
obtained low-MW CS had a MW of about 50 kDa (FIG. 1a). In a
preferred embodiment, the low molecular weight chitosan has a
molecular weight of less than about 40 kDa, but above 10 kDa. Other
forms of chitosan may also be applicable, including chitin,
chitosan oligosaccharides, and derivatives thereof.
[0088] It was observed that the obtained low-MW CS can be readily
dissolved in an aqueous solution at pH 6.0, while that before
depolymerization needs to be dissolved in an acetic acid solution
with a pH value about 4.0. Additionally, it was found that with the
low-MW CS, the prepared nanoparticles had a significantly smaller
size with a narrower distribution than their counterparts prepared
with the high-MW (also known as standard-MW) CS (before
depolymerization), due to its lower viscosity. As an example, upon
adding a 0.10% .gamma.-PGA aqueous solution into a 0.20% high-MW CS
solution (viscosity 5.73.+-.0.08 cp, measured by a viscometer), the
mean particle size of the prepared nanoparticles was 878.3.+-.28.4
nm with a polydispersity index of 1.0, whereas adding a 0.10%
.gamma.-PGA aqueous solution into the low-MW CS solution (viscosity
1.29.+-.0.02 cp) formed nanoparticles with a mean particle size of
218.1.+-.4.1 nm with a polydispersity index of 0.3 (n=5).
Example No. 3
Production and Purification of .gamma.-PGA
[0089] .gamma.-PGA was produced by Bacillus licheniformis (ATCC
9945, Bioresources Collection and Research Center, Hsinchu, Taiwan)
as per a method reported by Yoon et al. with slight modifications
(Biotechnol. Lett. 2000; 22:585-588). Highly mucoid colonies (ATCC
9945a) were selected from Bacillus licheniformis (ATCC 9945)
cultured on the E medium (ingredients comprising L-glutamic acid,
20.0 g/l; citric acid, 12.0 g/l; glycerol, 80.0 g/l; NH.sub.4Cl,
7.0 g/l; K.sub.2HPO.sub.4, 0.5 g/l; MgSO.sub.4.7H.sub.2O, 0.5 g/l;
FeCl.sub.3.6H.sub.2O, 0.04 g/l; CaCl.sub.2.2H.sub.2O, 0.15 g/l;
MnSO.sub.4.H.sub.2O, 0.104 g/l, pH 6.5) agar plates at 37.degree.
C. for several times.
[0090] Subsequently, young mucoid colonies were transferred into 10
ml E medium and grown at 37.degree. C. in a shaking incubator at
250 rpm for 24 hours. Afterward, 500 .mu.l of culture broth was
mixed with 50 ml E medium and was transferred into a 2.5-1
jar-fermentor (KMJ-2B, Mituwa Co., Osaka, Japan) containing 950 ml
of E medium. Cells were cultured at 37.degree. C. The pH was
controlled at 6.5 by automatic feeding of 25% (v/v) NH.sub.4OH
and/or 2M HCl. The dissolved oxygen concentration was initially
controlled at 40% of air saturation by supplying air and by
controlling the agitation speed up to 1000 rpm.
[0091] After 40 hours, cells were separated from the culture broth
by centrifugation for 20 minutes at 12,000.times.g at 4.degree. C.
The supernatant containing .gamma.-PGA was poured into 4 volumes of
methanol and left overnight with gentle stirring. The resulting
precipitate containing crude .gamma.-PGA was collected by
centrifugation for 40 minutes at 12,000.times.g at 4.degree. C. and
then was dissolved in deionized water to remove insoluble
impurities by centrifugation for 20 minutes at 24,000.times.g at
4.degree. C. The aqueous .gamma.-PGA solution was desalted by
dialysis (MWCO: 100,000, Spectrum Laboratories, Inc., Laguna Hills,
Calif.) against distilled water for 12 hours with water exchanges
several times, and finally was lyophilized to obtain pure
.gamma.-PGA.
[0092] The purified .gamma.-PGA was verified by the proton nuclear
magnetic resonance (.sup.1H-NMR) and the FT-IR analyses. Analysis
of .sup.1H-NMR was conducted on an NMR spectrometer (Varian
Unityionva 500 NMR Spectrometer, MO) using DMSO-d.sub.6 at 2.49 ppm
as an internal reference. Test samples used for the FT-IR analysis
first were dried and ground into a powder form. The powder then was
mixed with KBr (1:100) and pressed into a disk. Analysis was
performed on an FT-IR spectrometer (Perkin Elmer Spectrum RX1 FT-IR
System, Buckinghamshire, England). The samples were scanned from
400-4000 cm.sup.-1. The average molecular weight of the purified
.gamma.-PGA was determined by the same GPC system as described
before. Polyethylene glycol (molecular weights of 106-22,000) and
polyethylene oxide (molecular weights of 20,000-1,000,000, PL
Laboratories) standards were used to construct a calibration curve.
The mobile phase contained 0.01M NaH.sub.2PO.sub.4 and 0.2 M
NaNO.sub.3 and was brought to a pH of 7.0.
[0093] The purified .gamma.-PGA obtained from fermentation was
analyzed by GPC, .sup.1H-NMR, and FT-IR. As analyzed by GPC (FIG.
1b), the purified .gamma.-PGA had a MW of about 160 kDa. In the
FT-IR spectrum (FIG. 2a), a characteristic peak at 1615 cm.sup.-1
for the associated carboxylic acid salt (-COO.sup.- antisymmetric
stretch) on .gamma.-PGA was observed. The characteristic absorption
due to C.dbd.O in secondary amides (amide 1 band) was overlapped by
the characteristic peak of --COO.sup.-. Additionally, the
characteristic peak observed at 3400 cm.sup.-1 was the N--H stretch
of .gamma.-PGA. In the .sup.1H-NMR spectrum (FIG. 2b), six chief
signals were observed at 1.73 and 1.94 ppm (.beta.-CH.sub.2), 2.19
ppm (.gamma.-CH.sub.2), 4.14 ppm (.alpha.-CH), 8.15 ppm (amide),
and 12.58 ppm (COON). These results indicated that the observed
FT-IR and .sup.1H-NMR spectra correspond well to those expected for
.gamma.-PGA. Additionally, the fermented product after purification
showed no detected macromolecular impurities by the .gamma.H-NMR
analysis, suggesting that the obtained white power of .gamma.-PGA
is highly pure.
Example No. 4
Preparation of the CS-.gamma.-PGA Nanoparticles
[0094] Nanoparticles were obtained upon addition of .gamma.-PGA
aqueous solution (pH 7.4, 2 ml), using a pipette (0.5-5 ml,
PLASTIBRAND.RTM., BrandTech Scientific Inc., Germany), into a
low-MW CS aqueous solution (pH 6.0, 10 ml) at varying
concentrations (0.01%, 0.05%, 0.10%, 0.15%, or 0.20% by w/v) under
magnetic stirring at room temperature. Nanoparticles were collected
by ultracentrifugation at 38,000 rpm for 1 hour. Supernatants were
discarded and nanoparticles were resuspended in deionized water for
further studies. FT-IR was used to analyze peak variations of amino
groups of low-MW CS and carboxylic acid salts of .gamma.-PGA in the
CS-.gamma.-PGA nanoparticles.
[0095] As stated, nanoparticles were obtained instantaneously upon
addition of a .gamma.-PGA aqueous solution (pH 7.4) into a low-MW
CS aqueous solution (pH 6.0) under magnetic stirring at room
temperature. FIG. 3 shows the FT-IR spectra of the low-MW CS and
the CS-.gamma.-PGA nanoparticles. As shown in the spectrum of CS,
the characteristic peak observed at 1563 cm.sup.-1 was the
protonated amino group (--NH.sub.3.sup.+ deformation) on CS. In the
spectrum of CS-.gamma.-PGA complex, the characteristic peak at 1615
cm.sup.-1 for --COO.sup.- on .gamma.-PGA disappeared and a new peak
at 1586 cm.sup.-1 appeared, while the characteristic peak of
--NH.sub.3.sup.+ deformation on CS at 1563 cm.sup.-1 shifted to
1555 cm.sup.-1. These observations are attributed to the
electrostatic interaction between the negatively charged carboxylic
acid salts (--COO.sup.-) on .gamma.-PGA and the positively charged
amino groups (--NH.sub.3.sup.+) on CS. The electrostatic
interaction between the two polyelectrolytes (.gamma.-PGA and CS)
instantaneously induced the formation of long hydrophobic segments
at least segments with a high density of neutral ion-pairs), and
thus resulted in highly neutralized complexes that segregated into
colloidal nanoparticles.
Example No. 5
Characterization of the CS-.gamma.-PGA Nanoparticles
[0096] The morphological examination of the CS-.gamma.-PGA
nanoparticles was performed by TEM (transmission electron
microscopy) and AFM (atomic force microscopy). The TEM sample was
prepared by placing a drop of the nanoparticle solution onto a 400
mesh copper grid coated with carbon. About 2 minutes after
deposition, the grid was tapped with a filter paper to remove
surface water and positively stained by using an alkaline bismuth
solution. The AFM sample was prepared by casting a drop of the
nanoparticle solution on a slide glass and then dried in vacuum.
The size distribution and zeta potential of the prepared
nanoparticles were measured using a Zetasizer (3000HS, Malvern
Instruments Ltd., Worcestershire, UK).
[0097] During storage, aggregation of nanoparticles may occur and
thus leads to losing their structural integrity or forming
precipitation of nanoparticles. Therefore, the stability of
nanoparticles during storage must be evaluated. In the stability
study, the prepared nanoparticles suspended in deionized water (1
mg/ml) were stored at 4.degree. C. and their particle sizes and
zeta potential values were monitored by the same Zetasizer as
mentioned earlier during storage.
[0098] In the preparation of nanoparticles, samples were visually
analyzed and three distinct solution systems were identified: clear
solution, opalescent suspension, and solution with precipitation of
aggregates. Examined by the Zetasizer, nanoparticles were found in
the clear solution and the opalescent suspension rather than in the
solution with precipitation of aggregates.
[0099] The particle sizes and the zeta potential values of
CS-.gamma.-PGA nanoparticles, prepared at varying concentrations of
.gamma.-PGA and CS, were determined and the results are shown in
Tables 1a and 1b. It was found that the particle size and the zeta
potential value of the prepared nanoparticles were mainly
determined by the relative amount of the local concentration of
.gamma.-PGA in the added solution to the surrounding concentration
of CS in the sink solution. At a fixed concentration of CS, an
increase in the .gamma.-PGA concentration allowed .gamma.-PGA
molecules interacting with more CS molecules, and thus formed a
lager size of nanoparticles (Table 1a, p<0.05). When the amount
of CS molecules exceeded that of local .gamma.-PGA molecules, some
of the excessive CS molecules were entangled onto the surfaces of
CS-.gamma.-PGA nanoparticles.
[0100] Thus, the resulting nanoparticles may display a structure of
a neutral polyelectrolyte-complex core surrounded by a positively
charged CS shell (Table 1b) ensuring the colloidal stabilization
(Langmuir. 2004; 20:7766-7778). In contrast, as the amount of local
.gamma.-PGA molecules sufficiently exceeded that of surrounding CS
molecules, the formed nanoparticles had .gamma.-PGA exposed on the
surfaces and thus had a negative charge of zeta potential.
Therefore, the particle size and the zeta potential value of the
prepared CS-.gamma.-PGA nanoparticles can be controlled by their
constituted compositions. The results obtained by the TEM and AFM
examinations showed that the morphology of the prepared
nanoparticles was spherical in shape with a smooth surface (FIGS.
4a and 4b). Some aspects of the invention relate to nanoparticles
having a mean particle size between about 50 and 400 nanometers,
preferably between about 100 and 300 nanometers, and most
preferably between about 100 and 200 nanometers. The morphology of
the nanoparticles shows spherical in shape with a smooth surface at
any pH between 2.5 and 6.6. In one embodiment, the stability of the
nanoparticles of the present invention at a low pH around 2.5
enables the nanoparticles to be intact when exposed to the gastric
acidic medium.
TABLE-US-00001 TABLE 1a Effects of concentrations of .gamma.-PGA
and CS on the particle sizes of the prepared CS-.gamma.-PGA
nanoparticles Mean Particle Size (nm, n = 5) CS .gamma.-PGA
0.01%.sup.a) 0.05% 0.10% 0.15% 0.20% 0.01%.sup.b) 79.0 .+-. 3.0
103.1 .+-. 4.6 96.7 .+-. 1.9 103.6 .+-. 1.9 140.5 .+-. 2.0 0.05%
157.4 .+-. 1.7 120.8 .+-. 3.9 144.5 .+-. 2.4 106.2 .+-. 3.8 165.4
.+-. 1.7 0.10% 202.2 .+-. 3.1 232.6 .+-. 1.2 161.0 .+-. 1.8 143.7
.+-. 2.7 218.1 .+-. 4.1 0.15% 277.7 .+-. 3.2 264.9 .+-. 2.1 188.6
.+-. 2.9 178.0 .+-. 2.2 301.1 .+-. 6.4 0.20% 284.1 .+-. 2.1 402.2
.+-. 4.0 .tangle-solidup. 225.5 .+-. 3.1 365.5 .+-. 5.1
.sup.a)concentration of CS (by w/v) .sup.b)concentration of
.gamma.-PGA (by w/v) .tangle-solidup. precipitation of aggregates
was observed
TABLE-US-00002 TABLE 1b Effects of concentrations of .gamma.-PGA
and CS on the zeta potential values of the prepared CS-.gamma.-PGA
nanoparticles. Zeta Potential (mV, n = 5) CS .gamma.-PGA
0.01%.sup.a) 0.05% 0.10% 0.15% 0.20% 0.01%.sup.b) 15.4 .+-. 0.3
22.8 .+-. 0.5 19.8 .+-. 1.5 16.5 .+-. 1.4 17.2 .+-. 1.6 0.05% -32.7
.+-. 0.7 23.7 .+-. 1.7 27.6 .+-. 0.7 20.3 .+-. 0.8 19.2 .+-. 0.6
0.10% -33.1 .+-. 1.3 21.1 .+-. 1.6 20.3 .+-. 1.1 23.6 .+-. 0.9 24.7
.+-. 1.2 0.15% -33.2 .+-. 2.1 -21.9 .+-. 2.0 19.2 .+-. 0.4 16.9
.+-. 1.7 19.8 .+-. 0.3 0.20% -34.5 .+-. 0.5 -34.6 .+-. 0.3
.tangle-solidup. 14.6 .+-. 0.7 16.3 .+-. 0.7 .sup.a)concentration
of CS (by w/v) .sup.b)concentration of .gamma.-PGA (by w/v)
.tangle-solidup. precipitation of aggregates was observed
[0101] Two representative groups of the prepared nanoparticles were
selected for the stability study: one with a positive surface
charge (0.10% .gamma.-PGA:0.20% CS) and the other with a negative
surface charge (0.10% .gamma.-PGA:0.01% CS). FIG. 5 shows changes
in particle size (.box-solid., mean diameter) and zeta potential (
) of (a) the CS-.gamma.-PGA nanoparticles (0.10% .gamma.-PGA:0.20%
CS) and (b) the CS-.gamma.-PGA nanoparticles (0.10%
.gamma.-PGA:0.01% CS) during storage for up to 6 weeks. It was
found that neither aggregation nor precipitation of nanoparticles
was observed during storage for up to 6 weeks, as a result of the
electrostatic repulsion between the positively charged
CS-.gamma.-PGA nanoparticles (for the former group) or the
negatively charged CS-.gamma.-PGA nanoparticles (for the latter
group).
[0102] Additionally, changes in particle size and zeta potential of
the nanoparticles were minimal for both studied groups (FIGS. 5a
and 5b). These results demonstrated that the prepared nanoparticles
suspended in deionized water were stable during storage.
[0103] In a further study, NPs were self-assembled instantaneously
upon addition of an aqueous .gamma.-PGA into an aqueous TMC
(N-trimethyl chitosan) having a TMC/.gamma.-PGA weight ratio of 6:1
under magnetic stirring at room temperature. Other chitosan
derivative, such as mono-N-carboxymethyl chitosan (MCC), has also
been useful in self-assembled nanoparticle formation. The chemical
formulas of chitosan, N-trimethyl chitosan, and MCC are shown
below:
##STR00001##
[0104] Furthermore, other chitosan derivative, such as NPCS, has
also been useful in self-assembled nanoparticle formation.
N-palmitoyl CS (NPCS) is made of a hydrophobic palmitoyl group
conjugated onto the free amine groups of CS. NPCS is a comblike
polyelectrolyte characterized by the presence of alternating
charges (protonated amine groups) and hydrophobic side chains.
[0105] The amount of positively charged TMC significantly exceeded
that of negatively charged .gamma.-PGA; some of excessive TMC
molecules were entangled onto the surfaces of NPs, thus displaying
a positive surface charge (Table 2). The degree of quaternization
on TMC had little effects on the mean particle size and zeta
potential of NPs. In Table 2, TMC25, TMC40 and TMC55 indicate an
N-trimethyl chitosan with a degree of quaternization of about 25,
40, and 50%, respectively.
Example No. 6
pH-Responsive Characteristics of NPs
[0106] The stomach pH is about 1.0 to 2.0 in the presence of food,
while the fasting pH of the stomach is 2.5-3.7. The pH values in
the duodenum and the jejunum and proximal ileum are 6.0-6.6 and
6.6-7.0, respectively, while the mean pH in the distal ileum and in
the body fluid at intercellular spaces between enterocytes is about
7.4. Therefore, characterization of test NPs in response to
distinct pH environments must be investigated. TMC is a positively
charged polymer and has been used as an intestinal permeation
enhancer, and its mechanism of opening tight junctions is similar
to that of protonated CS. Additionally, TMC showed no indication of
epithelial damage or cytotoxicity. The basic mechanism of the
present disclosure is that the orally administered NPs with
excessive mucoadhesive TMC on their surfaces may adhere and
infiltrate into the mucus of the intestinal tract, and then mediate
transiently opening the tight junctions between enterocytes. It is
known that the tight junctions opened by absorption enhancers are
less than 20 nm in width. Consequently, the NPs infiltrated into
the mucus must become unstable (swelling or disintegration); thus
their loaded insulin can be released and permeated through the
paracellular pathway to the bloodstream.
TABLE-US-00003 TABLE 2 Mean particle sizes, zeta potential values
and polydispersity indices of nanoparticles (NPs) self-assembled by
TMC polymers with different degrees of quaternization and
.gamma.-PGA (n = 5 batches). Mean Particle Zeta Potential
Polydispersity Size (nm) (mV) Index CS/.gamma.-PGA NPs 104.1 .+-.
1.2 36.2 .+-. 2.5 0.11 .+-. 0.02 TMC25/.gamma.-PGA NPs 101.3 .+-.
3.1 30.9 .+-. 2.1 0.13 .+-. 0.04 TMC40/.gamma.-PGA NPs 106.3 .+-.
2.3 32.3 .+-. 2.1 0.15 .+-. 0.14 TMC55/.gamma.-PGA NPs 114.6 .+-.
2.3 30.6 .+-. 3.8 0.12 .+-. 0.03 TMC: N-trimethyl chitosan; CS:
chitosan; .gamma.-PGA: poly(.gamma.-glutamic acid).
[0107] It is known that the pKa values of CS (amine groups) and
.gamma.-PGA (carboxylic groups) are 6.5 and 2.9, respectively. In
the study, NPs were prepared in DI water (pH 6.0). At pH 6.0, CS
(TMC25) and .gamma.-PGA were ionized. The ionized CS (TMC25) and
.gamma.-PGA could form polyelectrolyte complexes, which resulted in
a matrix structure with a spherical shape. At pH 1.2-2.0, most
carboxylic groups on .gamma.-PGA were in the form of --COOH. Hence,
there was little electrostatic interaction between CS (TMC25) and
.gamma.-PGA; thus NPs became disintegrated. Similarly, at pH values
above 6.6, the free amine groups on CS (TMC25) were deprotonated;
thus leading to the disintegration of NPs. This might limit the
efficacy of drug delivery and absorption in the small
intestine.
[0108] With increasing the degree of quaternization on TMC (TMC40
and TMC55), the stability of NPs in the pH range of 6.6-7.4
increased significantly. However, the swelling of TMC55/.gamma.-PGA
NPs at pH 7.4 was minimal (due to the highly quaternized TMC55),
which might limit the release of loaded drugs. In contrast,
TMC40/.gamma.-PGA NPs swelled significantly with increasing the pH
value. TMC40/.gamma.-PGA NPs (or fragments) still retained a
positive surface charge with a zeta potential value of 17.3 mV at
pH 7.4. Thus, TMC40/.gamma.-PGA/drug NPs had superior stability in
a broader pH range to CS/.gamma.-PGA/drug NPs. In one embodiment,
at around body fluid pH of about 7.4, the bioactive nanoparticles
of the present invention may appear to be in configuration of
chitosan-shelled fragments or chitosan-containing fragments. At
least a portion of the surface of the chitosan-shelled fragments or
chitosan-containing fragments from the bioactive nanoparticles of
the present invention shows positive zeta potential
characteristics.
[0109] The results of molecular dynamic simulations showed that the
molecular chains of TMC40 and .gamma.-PGA in their self-assembled
complex were tightly entangled to each other at pH 6.0. The surface
of the complex was dominated by TMC40 molecules. Relaxations of
TMC40 and .gamma.-PGA molecular chains at pH 2.5 resulted in a
moderate swelling of the TMC40/.gamma.-PGA complex, while its
surface was still dominated by the positively charged TMC
molecules, thus retaining a positive surface charge. Similarly,
relaxations of TMC40 and .gamma.-PGA molecular chains at pH 7.4
resulted in a significant swelling of the TMC40/.gamma.-PGA
complex, while its surface was still dominated by the positively
charged TMC molecules, thus retaining a positive surface charge.
The swollen TMC40/.gamma.-PGA/drug nanoparticles tend to slightly
disintegrate so to form fragments consisting of
TMC40/.gamma.-PGA/drug with surface-dominated TMC40. The
TMC40/.gamma.-PGA/drug fragments with surface-dominated TMC40 would
adhere and infiltrate into the mucus of the epithelial membrane of
the blood-brain barrier, and then mediate transiently opening the
tight junctions between enterocytes.
Example No. 7
Caco-2 Cell Cultures and TEER Measurements
[0110] Caco-2 cells were seeded on the tissue-culture-treated
polycarbonate filters (diameter 24.5 mm, growth area 4.7 cm.sup.2)
in Costar Transwell 6 wells/plates (Corning Costar Corp., NY) at a
seeding density of 3.times.10.sup.5 cells/insert. MEM (pH 7.4)
supplemented with 20% FBS, 1% NEAA, and 40 .mu.g/ml
antibiotic-gentamicin was used as the culture medium, and added to
both the donor and acceptor compartments. The medium was replaced
every 48 hours for the first 6 days and every 24 hours thereafter.
The cultures were kept in an atmosphere of 95% air and 5% CO, at
37.degree. C. and were used for the paracellular transport
experiments 18-21 days after seeding (TEER values in the range of
600-800 .OMEGA.cm.sup.2).
[0111] TEER values of the Caco-2 cell monolayers were monitored
with a Millicell.RTM.-Electrical Resistance System (Millipore
Corp., Bedford, Mass.) connected to a pair of chopstick electrodes.
To initiate the transport experiments, the culture media in the
donor and acceptor compartments were aspirated, and the cells were
rinsed twice with pre-warmed transport media (HBSS supplemented
with 25 mM glucose, pH 6.0). Following a 30-min equilibration with
the transport media at 37.degree. C., the cells were incubated for
2 hours with 2 ml transport media containing 0.5 ml test
nanoparticle solutions (0.2 mg/ml) at 37.degree. C. Subsequently,
solutions of nanoparticles were carefully removed and cells were
washed three times with HBSS and replaced by fresh culture media.
The TEER was measured for another 20 hours to study reversibility
of the effect of test nanoparticles on Caco-2 cell monolayers.
[0112] The intercellular tight junction is one of the major
barriers to the paracellular transport of macromolecules (J.
Control. Release 1996; 39:131-138; J. Control. Release 1998;
51:35-46). Trans-epithelial ion transport is contemplated to be a
good indication of the tightness of the junctions between cells and
was evaluated by measuring TEER of Caco-2 cell monolayers in the
study. It was reported that the measurement of TEER can be used to
predict the paracellular transport of hydrophilic molecules (Eur.
J. Pharm. Biopharm. 2004; 58:225-235). When the tight junctions
open, the TEER value will be reduced due to the water and ion
passage through the paracellular route. Caco-2 cell monolayers have
been widely used as an in vitro model to evaluate the intestinal
paracellular permeability of macromolecules.
[0113] Effects of the prepared CS-.gamma.-PGA nanoparticles on the
TEER values of Caco-2 cell monolayers are shown in FIG. 6. As
shown, the prepared nanoparticles with a positive surface charge
(CS dominated on the surface, 0.01% .gamma.-PGA:0.05% CS, 0.10%
.gamma.-PGA:0.2% CS, and 0.20% .gamma.-PGA:0.20% CS) were able to
reduce the values of TEER of Caco-2 cell monolayers significantly
(p<0.05). After a 2-hour incubation with these nanoparticles,
the TEER values of Caco-2 cell monolayers were reduced to about 50%
of their initial values as compared to the control group (without
addition of nanoparticles in the transport media). This indicated
that the nanoparticles with CS dominated on the surfaces could
effectively open or loosen the tight junctions between Caco-2
cells, resulting in a decrease in the TEER values. It was reported
that interaction of the positively charged amino groups of CS with
the negatively charged sites on cell surfaces and tight junctions
induces a redistribution of F-actin and the tight junction's
protein ZO-1, which accompanies the increased paracellular
permeability. It is suggested that an interaction between chitosan
and the tight junction protein ZO-1, leads to its translocation to
the cytoskeleton.
[0114] After removal of the incubated nanoparticles, a gradual
increase in TEER values was noticed. This phenomenon indicated that
the intercellular tight junctions of Caco-2 cell monolayers started
to recover gradually; however, the TEER values did not recover to
their initial values (FIG. 6). Kotze et al. reported that complete
removal of a CS-derived polymer, without damaging the cultured
cells, was difficult due to the highly adhesive feature of CS
(Pharm. Res. 1997; 14:1197-1202). This might be the reason why the
TEER values did not recover to their initial values. In contrast,
the TEER values of Caco-2 cell monolayers incubated with the
nanoparticles with a negative surface charge (.gamma.-PGA dominated
on the surface, 0.10% .gamma.-PGA:0.01% CS and 0.20%
.gamma.-PGA:0.01% CS, FIG. 6) showed no significant differences as
compared to the control group (p>0.05). This indicated that
.gamma.-PGA does not have any effects on the opening of the
intercellular tight junctions.
[0115] Transformation of chitosan into nanoparticles significantly
promoted its association with the Caco-2 cell monolayers. It also
enabled the polymer to be internalized by the cells through
clathrin-dependent endocytosis pathway (Pharmaceutical Research
2003; 20:1812-1819). Endocytosis is a process where cells absorb
material (molecules such as proteins) from the outside by engulfing
it with their cell membrane.
[0116] FIG. 8 shows an illustrative protein transport mechanism
through a cellular layer, including transcellular transport and
paracellular transport. FIG. 9 shows a schematic illustration of a
paracellular transport mechanism. The transcellular protein or
peptide transport may be an active transport or a passive transport
mode whereas the paracellular transport is basically a passive
mode. Ward et al. reported and reviewed current knowledge regarding
the physiological regulation of tight junctions and paracellular
permeability (PSTT 2000; 3:346-358). Chitosan as nanoparticle
vehicles for oral delivery of protein drugs avoids the enzymatic
inactivation in the gastrointestinal conduit. The chitosan
component of the present nanoparticles has a special feature of
adhering to the mucosal surface and transiently opening the tight
junctions between epithelial cells; that is, loosening the
tightness of the tight junctions.
[0117] FIG. 9(A) shows that after feeding nanoparticles (NPs)
orally, NPs adhere and infiltrate into the mucus layer of the
epithelial cells. FIG. 9(B) illustrates that the infiltrated NPs
transiently and reversibly loosen tight junctions (TJs) while
becoming unstable and disintegrated to release insulin or entrapped
agent. FIG. 9(c) shows that the released insulin or API permeates
through the paracellular pathway into the blood stream. Here, a
complexone (EGTA, EDTA, EGTA, and the like) has been shown to
transiently and reversibly widen/open the tight junctions by
removing Ca.sup.2+ ions from the basolateral side of epithelial
cells. Alternately, chitosan (CS), a nontoxic, soft-tissue
compatible, cationic polysaccharide has special features of
adhering to the mucosal surface; CS is able to transiently and
reversibly widen/loosen TJs between epithelial cells. The TJ width
in the small intestine has been demonstrated to be less than 1 nm.
It is also known that TJs `opened` by absorption enhancers are less
than 20 nm wide (Nanotechnology 2007; 18:1-11). The term "opened"
herein means that any substance less than 20 nm in the
close-proximity might have the chance to pass through. TJs
constitute the principal barrier to passive movement of fluid,
electrolytes, macromolecules and cells through the paracellular
pathway.
[0118] It was suggested that the electrostatic interaction between
the positively charged CS and the negatively charged sites of ZO-1
proteins on cell surfaces at TJ induces a redistribution of
cellular F-actin and ZO-1's translocation to the cytoskeleton,
leading to an increase in paracellular permeability. As evidenced
in FIG. 9, after adhering and infiltrating into the mucus layer of
the duodenum, the orally administered nanoparticles may degrade due
to the presence of distinct digestive enzymes in the intestinal
fluids. Additionally, the pH environment may become neutral while
the nanoparticles were infiltrating into the mucosa layer and
approaching the intestinal epithelial cells. This further leads to
the collapse of nanoparticles due to the change in the exposed pH
environment. The dissociated CS from the degraded/collapsed
nanoparticles was then able to interact and modulate the function
of ZO-1 proteins between epithelial cells (Nanotechnology 2007;
18:1-11). ZO-1 proteins are thought to be a linkage molecule
between occludin and F-actin cytoskeleton and play important roles
in the rearrangement of cell-cell contacts at TJs.
Example No. 8
fCS-.gamma.-PGA Nanoparticle Preparation and CLSM Visualization
[0119] Fluorescence (FITC)-labeled CS-.gamma.-PGA (fCS-.gamma.-PGA)
nanoparticles were prepared for the confocal laser scanning
microscopy (CLSM) study. The nanoparticles of the present invention
display a structure of a neutral polyelectrolyte-complex core
surrounded by a positively charged chitosan shell. Synthesis of the
FITC-labeled low-MW CS (fCS) was based on the reaction between the
isothiocyanate group of FITC and the primary amino groups of CS as
reported in the literature (Pharm. Res. 2003; 20:1812-1819).
Briefly, 100 mg of FITC in 150 ml of dehydrated methanol were added
to 100 ml of 1% low-MW CS in 0.1M acetic acid. After 3 hours of
reaction in the dark at ambient conditions, fCS was precipitated by
raising the pH to about 8-9 with 0.5M NaOH. To remove the
unconjugated FITC, the precipitate was subjected to repeated cycles
of washing and centrifugation (40,000.times.g for 10 min) until no
fluorescence was detected in the supernatant. The fCS dissolved in
80 ml of 0.1M acetic acid was then dialyzed for 3 days in the dark
against 5 liters of distilled water, with water replaced on a daily
basis. The resultant fCS was lyophilized in a freeze dryer. The
fCS-.gamma.-PGA nanoparticles were prepared as per the procedure
described in Example No. 4.
[0120] Subsequently, the transport medium containing
fCS-.gamma.-PGA nanoparticles (0.2 mg/ml) was introduced into the
donor compartment of Caco-2 cells, which were pre-cultured on the
transwell for 18-21 days. The experimental temperature was
maintained at 37.degree. C. by a temperature control system (DH-35
Culture Dish Heater, Warner Instruments Inc., Hamden, Conn.). After
incubation for specific time intervals, test samples were
aspirated. The cells were then washed twice with pre-warmed PBS
solution before they were fixed in 3.7% paraformaldehyde (Phar.
Res. 2003; 20:1812-1819). Cells were examined under an inversed
CLSM (TCS SL, Leica, Germany). The fluorescence images were
observed using an argon laser (excitation at 488 nm, emission
collected at a range of 510-540 nm).
[0121] CLSM was used to visualize the transport of the
fluorescence-labeled CS-.gamma.-PGA (fCS-.gamma.-PGA) nanoparticles
across the Caco-2 cell monolayers. This non-invasive method allows
for optical sectioning and imaging of the transport pathways across
the Caco-2 cell monolayers, without disrupting their structures.
FIGS. 7a and 7b show the fluorescence images of 4 optical sections
of a Caco-2 cell monolayer that had been incubated with the
fCS-.gamma.-PGA nanoparticles having a positive surface charge
(0.10% .gamma.-PGA:0.20% CS, zeta potential: about 21 mV) for 20
and 60 min, respectively. As shown, after 20 min of incubation with
the nanoparticles, intense fluorescence signals at intercellular
spaces were observed at depths of 0 and 5 .mu.m from the apical
(upper) surface of the cell monolayer. The intensity of
fluorescence became weaker at levels deeper than 10 .mu.m from the
apical surface of the cell monolayer and was almost absent at
depths .gtoreq.15 .mu.m (FIG. 7a).
[0122] After 60 minutes of incubation with the nanoparticles, the
intensity of fluorescence observed at intercellular spaces was
stronger and appeared at a deeper level than those observed at 20
min after incubation. These observations confirmed with our TEER
results that the nanoparticles with a positive surface charge (CS
dominated on the surface) were able to open the tight junctions
between Caco-2 cells and allowed transport of the nanoparticles by
passive diffusion via the paracellular pathways.
Example No. 9
In Vivo Study with Fluorescence-Labeled Nanoparticles
[0123] Fluorescence (FITC)-labeled CS-.gamma.-PGA (fCS-.gamma.-PGA)
nanoparticles were prepared for the confocal laser scanning
microscopy (CLSM) study. After feeding rats with fCS-.gamma.-PGA
nanoparticles, the rats are sacrificed at a pre-determined time and
the intestine is isolated for CLSM examination. The fluorescence
images of the nanoparticles were clearly observed by CLSM that
penetrates through the mouse intestine at appropriate time and at
various depths from the inner surface toward the exterior surface
of the intestine, including duodenum, jejunum, and ileum.
Example No. 10
Insulin Loading Capacity in Nanoparticles
[0124] Fluorescence (FITC)-labeled .gamma.-PGA was added into
chitosan solution to prepare fluorescence (FITC)-labeled,
insulin-loaded CS-.gamma.-PGA nanoparticles for in vivo animal
study with confocal laser scanning microscopy (CLSM) assessment and
bioactivity analysis. The insulin-loaded CS-.gamma.-PGA
nanoparticles are by using the ionic-gelation method upon addition
of insulin mixed with .gamma.-PGA solution into CS solution,
followed by magnetic stirring in a container.
[0125] Model insulin used in the experiment and disclosed herein is
obtained from bovine pancreas (Sigma-Aldrich, St. Louis, Mo.),
having a molecular formula of
C.sub.254H.sub.377N.sub.65O.sub.75S.sub.6 with a molecular weight
of about 5733.5 and an activity of .gtoreq.27 USP units/mg. The
insulin contains two-chain polypeptide hormone produced by the
.beta.-cells of pancreatic islets. The .alpha. and .beta. chains
are joined by two interchain disulfide bonds. Insulin regulates the
cellular uptake, utilization, and storage of glucose, amino acids,
and fatty acids and inhibits the breakdown of glycogen, protein,
and fat. The insulin from Sigma-Aldrich contains about 0.5% zinc.
Separately, insulin can be obtained from other sources, such as
human insulin solution that is chemically defined, recombinant from
Saccharomyces cerevisiae. Some aspects of the invention relate to
nanoparticles with insulin in the core, wherein the insulin may
contain intermediate-acting, regular insulin, rapid-acting insulin,
sustained-acting insulin that provides slower onset and longer
duration of activity than regular insulin, or combinations
thereof.
[0126] Examples of insulin or insulin analog products include, but
not limited to, Humulin.RTM. (by Eli Lilly), Humalog.RTM. (by Eli
Lilly) and Lantus.RTM. (by Aventis), and Novolog.RTM. Mix70/30 (by
Novo Nordisk). Humalog (insulin lispro, rDNA origin) is a human
insulin analog that is a rapid-acting, parenteral blood
glucose-lowering agent. Chemically, it is Lys(B28), Pro(B29) human
insulin analog, created when the amino acids at positions 28 and 29
on the insulin B-chain are reversed. Humalog is synthesized in a
special non-pathogenic laboratory strain of Escherichia coli
bacteria that has been genetically altered by the addition of the
gene for insulin lispro. Humalog has the empirical formula
C.sub.257H.sub.383N.sub.65O.sub.77S.sub.6 and a molecular weight of
5808, identical to that of human insulin. The vials and cartridges
contain a sterile solution of Humalog for use as an injection.
Humalog injection consists of zinc-insulin lispro crystals
dissolved in a clear aqueous fluid. Each milliliter of Humalog
injection contains insulin lispro 100 Units, 16 mg glycerin, 1.88
mg dibasic sodium phosphate, 3.15 mg m-cresol, zinc oxide content
adjusted to provide 0.0197 mg zinc ion, trace amounts of phenol,
and water for injection. Insulin lispro has a pH of 7.0-7.8.
Hydrochloric acid 10% and/or sodium hydroxide 10% may be added to
adjust pH.
[0127] Humulin is used by more than 4 million people with diabetes
around the world every day. Despite its name, this insulin does not
come from human beings. It is identical in chemical structure to
human insulin and is made in a factory using a chemical process
called recombinant DNA technology. Humulin L is an amorphous and
crystalline suspension of human insulin with a slower onset and a
longer duration of activity (up to 24 hours) than regular insulin.
Humulin U is a crystalline suspension of human insulin with zinc
providing a slower onset and a longer and less intense duration of
activity (up to 28 hours) than regular insulin or the
intermediate-acting insulins (NPH and Lente).
[0128] LANTUS.RTM. (insulin glargine [rDNA origin] injection) is a
sterile solution of insulin glargine for use as an injection.
Insulin glargine is a recombinant human insulin analog that is a
long-acting (up to 24-hour duration of action), parenteral
blood-glucose-lowering agent. LANTUS is produced by recombinant DNA
technology utilizing a non-pathogenic laboratory strain of
Escherichia coli (K12) as the production organism. Insulin glargine
differs from human insulin in that the amino acid asparagine at
position A21 is replaced by glycine and two arginines are added to
the C-terminus of the B-chain. Chemically, it is
21.sup.A-Gly-30.sup.Ba-L-Arg-30.sup.Bb-L-Arg-human insulin and has
the empirical formula C.sub.267H.sub.404N.sub.72O.sub.78S.sub.6 and
a molecular weight of 6063.
[0129] LANTUS consists of insulin glargine dissolved in a clear
aqueous fluid. Each milliliter of LANTUS (insulin glargine
injection) contains 100 IU (3.6378 mg) insulin glargine. Inactive
ingredients for the 10 mL vial are 30 mcg zinc, 2.7 mg m-cresol, 20
mg glycerol 85%, 20 mcg polysorbate 20, and water for injection.
Inactive ingredients for the 3 mL cartridge are 30 mcg zinc, 2.7 mg
m-cresol, 20 mg glycerol 85%, and water for injection. In 2006,
there were 11.4 million prescriptions of Lantus in the U.S. for
basal insulin maintenance.
[0130] Novolog.RTM. Mix70/30 (70% insulin aspart protamine
suspension and 30% insulin aspart injection [rDNA origin]) is a
human insulin analog suspension. Novolog.RTM. Mix70/30 is a blood
glucose-lowering agent with a rapid onset and an intermediate
duration of action. Insulin aspart is homologous with regular human
insulin with the exception of a single substitution of the amino
acid praline by aspartic acid in position B28, and is produced by
recombinant DNA technology utilizing Saccharomyces cerevisiae as
the production organism. Insulin aspart (Novolog) has the empirical
formula C.sub.256H.sub.381N.sub.65O.sub.79S.sub.6 and a molecular
weight of 5826. Novolog.RTM. Mix70/30 is a uniform, white sterile
suspension that contains zinc 19.6 .mu.g/ml and other
components.
[0131] The nanoparticles with two insulin concentrations are
prepared at a chitosan to .gamma.-PGA ratio of 0.75 mg/ml to 0.167
mg/ml. Their particle size and zeta potential are shown in Table 3
below.
TABLE-US-00004 TABLE 3 Insulin Conc. Mean Particle Size
Polydispersity Zeta Potential (mg/ml) (n = 5) (nm) Index (PI) (mV)
0* 145.6 .+-. 1.9 0.14 .+-. 0.01 +32.11 .+-. 1.61 0.042 185.1 .+-.
5.6 0.31 .+-. 0.05 +29.91 .+-. 1.02 0.083 198.4 .+-. 6.2 0.30 .+-.
0.09 +27.83 .+-. 1.22 *control reference without insulin
[0132] Further, their association efficiency of insulin and loading
capacity of insulin are analyzed, calculated and shown in FIGS. 11
and 12, according to the following formula:
Insulin Association Efficiency ( LE % ) = ( Total amount of insulin
- Insulin in supernatant ) Total amount of insulin .times. 100 %
##EQU00001## Loading Capacity ( LC ) = ( Total amount of insulin -
Insulin in supernatant ) Weight of recovered particles .times. 100
% ##EQU00001.2##
[0133] FIG. 11 shows loading capacity and association efficiency of
insulin in nanoparticles of chitosan and .gamma.-PGA, whereas FIG.
12 shows loading capacity and association efficiency of insulin in
nanoparticles of chitosan alone (in absence of .gamma.-PGA) as
reference. The data clearly demonstrates that both the insulin
loading capacity and insulin association efficiency are
statistically higher for the nanoparticles with .gamma.-PGA in the
core. The LE (40.about.55%) and LC (5.0.about.14.0%) of insulin for
CS-.gamma. PGA nanoparticles was obtained by using ionic-gelation
method upon addition of insulin mixed with .gamma.-PGA solution
into CS solution, followed by magnetic stirring for nanoparticle
separation.
[0134] In certain follow-up experiments, nanoparticles having a
pharmaceutical composition have been successfully prepared with a
negatively charged component comprised of .gamma.-PGA, .alpha.-PGA,
PGA derivatives, salts of PGA, heparin or heparin analog,
glycosaminoglycans, or alginate. The PGA derivatives of the present
invention may include, but not limited to, poly-.gamma.-glutamic
acid, poly-.alpha.-glutamic acid, poly-L-glutamic acid
(manufactured by Sigma-Aldrich, St. Louis, Mo.), poly-D-glutamic
acid, poly-L-.alpha.-glutamic acid, poly-.gamma.-D-glutamic acid,
poly-.gamma.-DL-glutamic acid, and PEG or PHEG derivatives of
polyglutamic acid, salts of the above-cited PGAs, and the like.
Some aspects of the invention relate to nanoparticles comprising a
shell component and a core component, wherein at least a portion of
the shell component comprises chitosan and wherein the core
component is comprised of a negatively charged compound that is
conjugated to chitosan, and a bioactive agent.
[0135] The nanoparticle of the present invention that contains at
least one bioactive agent is generally referred herein as
"bioactive nanoparticle" (also known as "therapeutic
nanoparticle"). Some aspects of the invention relate to an oral
dose of nanoparticles that effectively enhance intestinal or blood
brain paracellular transport comprising a negative component (such
as .gamma.-PGA, .alpha.-PGA, PGA derivatives, heparin, or alginate)
and at least one enzyme-resistant agent in the core, wherein the
negatively charged substrate is at least partially neutralized with
a portion of the positively charged chitosan, wherein the chitosan
dominates on a surface of the nanoparticles with positive charges.
In one embodiment, the enzyme-resistant agent is complexone, such
as diethylene triamine pentaacetic acid (DTPA) or ethylene diamine
tetraacetic acid (EDTA), which conjugates with the chitosan
substrate or the PGA substrate in the nanoparticle formulation.
[0136] Nanoparticles Loaded with DTPA
[0137] Some aspects of the invention relate to a pharmaceutical
composition of nanoparticle comprising chitosan, PGA-complexone
conjugate and a bioactive agent. In one embodiment, the
PGA-complexone conjugate may broadly include a conjugate with PGA
derivatives such as .gamma.-PGA, .alpha.-PGA, derivatives of PGA or
salts of PGA, whereas the complexone may cover DTPA (diethylene
triamine pentaacetic acid), EDTA (ethylene diamine tetra acetate),
IDA (iminodiacetic acid), NTA (nitrilotriacetic acid), EGTA
(ethylene glycol tetraacetic acid), BAPTA
(1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid), DOTA
(1,4,7,10-tetraazacyclododecane-N,N',N,N'-tetraacetic acid), NOTA
(2,2',2''-(1,4,7-triazonane-1,4,7-triyl)triacetic acid), and the
like. A polyamino carboxylic acid (complexone) is a compound
containing one or more nitrogen atoms connected through carbon
atoms to one or more carboxyl groups.
[0138] Diethylene triamine pentaacetic acid (DTPA) is a polyamino
carboxylic acid consisting of a diethylenetriamine backbone
modified with five carboxymethyl groups. The molecule can be viewed
as an expanded version of EDTA. DTPA is used as its conjugate base,
often undefined, which has a high affinity for metal cations. In
example, upon complexation to lanthanide and actinide ions, DTPA
exists as the pentaanionic form, i.e. all five carboxylic acid
groups are deprotonated. DTPA has a molecular formula of
C.sub.14H.sub.23N.sub.3O.sub.10 with molar mass 393.358 g/mole and
a chemical formula as:
##STR00002##
[0139] Currently, DTPA is approved by the U.S. Food and Drug
Administration (FDA) for chelation of three radioactive materials:
plutonium, americium, and curium. DTPA is the parent acid of an
octadentate ligand, diethylene triamine pentaacetate. In some
situations, all five acetate arms are not attached to the metal
ion. In one aspect of the present invention, DTPA has been
conjugated to .gamma.-PGA through hexanediamine
((.gamma.-PGA)-DTPA) as illustrated below:
##STR00003##
[0140] In one aspect of the invention, (.gamma.-PGA)-DTPA is one
species of the PGA-complexone conjugates used in the current
pharmaceutical composition of nanoparticles. The overall degree of
substitution of DTPA in (.gamma.-PGA)-DTPA conjugate is generally
in the range of about 1-70%, preferably in the range of about
5-40%, and most preferably in the range of about 10-30%. DTPA does
not build up in the body or cause long-term health effects.
[0141] Nanoparticles comprising chitosan, PGA-complexone conjugates
and at least one bioactive agent using the simple and mild
ionic-gelation process described herein has demonstrated the
desired paracellular transport efficacy with TEER measurements in
the Caco-2 cell cultures model as described in Example No. 7.
Example No. 11
Enzyme Inhibition Study with (.gamma.-PGA)-DTPA Conjugate
[0142] Brush border membrane bounded enzymes were used to simulate
a contacting membrane at the bottom of a donor compartment, wherein
the insulin-loaded medium (Krebs-Ringer buffer) in the donor
compartment was used as the starting material at time zero. Three
elements were used in this enzyme inhibition study to assess the
enzymatic degradation of insulin versus time by brush border
membrane bounded enzymes. They were (a) insulin 1 mg/ml as control;
(b) DTPA 5 mg/ml; and (c) (.gamma.-PGA)-DTPA 5 mg/ml. As shown in
FIG. 22, both DTPA and (.gamma.-PGA)-DTPA substantially protect or
maintain the insulin activity or viable content over the
experimental duration up to 2 hours. Some aspects of the present
invention provide a pharmaceutical composition of nanoparticles,
the nanoparticles comprising a shell portion that is dominated by
positively charged chitosan, a core portion that comprises
complexone and one negatively charged substrate, wherein the
substrate is PGA, wherein the negatively charged substrate is at
least partially neutralized with a portion of the positively
charged chitosan in the core portion, and at least one bioactive
agent loaded within the nanoparticles. In one embodiment, the PGA
is conjugated with the complexone to form PGA-complexion conjugates
within the nanoparticles.
[0143] Some aspects of the invention provide a method of enhancing
enzymatic resistance of a bioactive agent in oral administration by
encapsulating the bioactive agent in nanoparticles, wherein the
nanoparticles have a pharmaceutical formulation and/or composition
as described in this disclosure and in claims. In one embodiment,
the nanoparticles are further loaded with pharmaceutically
acceptable carrier, diluent, or excipient in tablets, pills,
capsules, and the like.
[0144] Some aspects of the invention relate to a dose of
nanoparticles that effectively enhance intestinal or blood brain
paracellular transport comprising a polyanionic component (such as
.gamma.-PGA, .alpha.-PGA, PGA-DTPA, PGA derivatives, heparin,
heparin analogs, low molecular weight heparin, glycosaminoglycans,
or alginate) in the core and low molecular weight chitosan in the
shell, wherein the chitosan dominates on a surface of the
nanoparticles with surface positive charges. In use, firstly,
encapsulate the Alzheimer's drug in the chitosan shell nanoparticle
as described herein, wherein the nanoparticle is partially
crosslinked (optionally) to enhance its biodurability. Then
intra-venously inject the nanoparticles, whereby the nanoparticles
pass to the brain in blood circulation. The chitosan shell of the
nanoparticles adheres to the surface adjacent the tight junction in
the brain. Thereafter, the chitosan nanoparticle opens the tight
junction, wherein the Alzheimer's drug is released after passing
the tight junction for therapeutic treatment. In one embodiment,
the nanoparticles are in a spherical shape having a mean particle
size of about 50 to 250 nanometers, preferably 150 nanometers to
250 nanometers.
[0145] In one example, intravenous administration of the
nanoparticles comprising chitosan shell substrate, polyanionic core
substrate and at least one bioactive agent for treating Alzheimer's
disease in a patient is typically performed with 10 mg to 40 mg of
active agent per day over a period of one month to one year. The
bioactive agent is selected from the group consisting of
donepezile, rivastignine, galantamine, and/or those trade-named
products, such as memantine hydrochloride (Axura.RTM. by Merz
Pharmaceuticals), donepezil hydrochloride (Aricept.RTM. by Eisai
Co. Ltd.), rivastigmine tartrate (Exelon.RTM. by Novartis),
galantamine hydrochloride (Reminyl.RTM. by Johnson & Johnson),
and tacrine hydrochloride (Cognex.RTM. by Parke Davis).
[0146] Some aspects of the invention relate to a nanoparticle with
a core substrate comprising polyglutamic acids such as water
soluble salt of polyglutamic acids (for example, ammonium salt) or
metal salts of polyglutamic acid (for example, lithium salt, sodium
salt, potassium salt, magnesium salt, and the like). In one
embodiment, the form of polyglutamic acid may be selected from the
group consisting of poly-.alpha.-glutamic acid,
poly-L-.alpha.-glutamic acid, poly-.gamma.-glutamic acid,
poly-D-glutamic acid, poly-.gamma.-D-glutamic acid,
poly-.gamma.-DL-glutamic acid, poly-L-glutamic acid (manufactured
by Sigma-Aldrich, St. Louis, Mo.), and PEG or PHEG derivatives of
polyglutamic acid. Alginate is generally non-biodegradable;
however, it is stipulated that an alginate particle with about
30-50 kDa molecular weight is kidney inert. Heparin with negatively
charged side-groups has a general chemical structure as shown
below:
##STR00004##
[0147] Some aspects of the invention relate to the negatively
charged glycosaminoglycans (GAGs) as the core substrate of the
present nanoparticles. GAGs may be used to complex with a
low-molecular-weight chitosan to form drug-carrier nanoparticles.
GAGs may also conjugate with the protein drugs as disclosed herein
to enhance the bonding efficiency of the core substrate in the
nanoparticles. Particularly, the negatively charged core substrate
(such as GAGs, heparin, PGA, alginate, and the like) of the
nanoparticles of the present invention may conjugate with
chondroitin sulfate, hyaluronic acid, PDGF-BB, BSA, EGF, MK, VEGF,
KGF, bFGF, aFGF, MK, PTN, etc.
[0148] Calceti et al. reported an in vivo evaluation of an oral
insulin-PEG delivery system (Eur J Pharma Sci 2004; 22:315-323).
Insulin-PEG was formulated into mucoadhesive tablets constituted by
the thiolated polymer poly(acrylic acid)-cysteine. The therapeutic
agent was sustained released from these tablets within 5 hours. In
vivo, by oral administration to diabetic mice, the glucose levels
were found to decrease significantly over the time. Further,
Krauland et al. reported another oral insulin delivery study of
thiolated chitosan-insulin tablets on non-diabetic rats (J.
Control. Release 2004, 95:547-555). The delivery tablets utilized
2-Iminothiolane covalently linked to chitosan to form chitosan-TBA
(chitosan-4-thiobutylamidine) conjugate. After oral administration
of chitosan-TBA-insulin tablets to non-diabetic conscious rats, the
blood glucose level decreased significantly for 24 hours;
supporting the observation of sustained insulin release of the
presently disclosed nanoparticles herein through intestinal
absorption. In a further report by Morcol et al. (Int. J. Pharm.
2004; 277:91-97), an oral delivery system comprising calcium
phosphate-PEG-insulin-casein particles displays a prolonged
hypoglycemic effect after oral administration to diabetic rats.
[0149] Pan et al. disclosed chitosan nanoparticles improving the
intestinal absorption of insulin in vivo (Int J Pharma 2002;
249:139-147) with insulin-chitosan nanoparticles at a particle size
of 250-400 nm, a polydispersity index smaller than 0.1, positively
charged and stable. After administering the insulin-chitosan
nanoparticles, it was found that the hypoglycemic was prolonged
with enhanced pharmacological bioavailability. Their data confirmed
our observation as shown in FIGS. 11 and 12; however, the insulin
loading capacity and insulin association efficiency of the present
invention are substantially higher for the chitosan-insulin
nanoparticles with .gamma.-PGA in the core as the core
substrate.
Example No. 12
Insulin Nanoparticle Stability
[0150] FIG. 13 shows the stability of insulin-loaded nanoparticles
of the present invention with an exemplary composition of CS 0.75
mg/ml, .gamma.-PGA 0.167 mg/ml, and insulin 0.083 mg/ml. The
prepared insulin-loaded nanoparticles suspended in deionized water
are stable during storage up to 40 days. First (in FIG. 13), the
insulin content in the nanoparticle storage solution maintains at
about a constant level of 9.5%. The nanoparticle stability is
further evidenced by the substantially constant particle size at
about 200 nm and substantially constant zeta potential of about +28
mV over the period of about 40 days. It is contemplated that the
insulin-containing nanoparticles of the present invention would
further maintain their biostability when formulated in a soft
gelcap or capsule configuration that further isolates the
nanoparticles from environmental effects, such as sunlight, heat,
air conditions, and the like. Some aspects of the invention provide
a gelcap pill or capsule containing a dosage of insulin
nanoparticles effective amount of the insulin to treat or manage
the diabetic patients, wherein the stability of the
insulin-containing nanoparticles is at least 40 days, preferably
more than 6 months, and most preferably more than a couple of
years.
[0151] By "effective amount of the insulin", it is meant that a
sufficient amount of insulin will be present in the dose to provide
for a desired therapeutic, prophylatic, or other biological effect
when the compositions are administered to a host in the single
dosage forms. The capsule of the present invention may preferably
comprise two-part telescoping gelatin capsules. Basically, the
capsules are made in two parts by dipping metal rods in molten
gelatin solution. The capsules are supplied as closed units to the
pharmaceutical manufacturer. Before use, the two halves are
separated, the capsule is filled with powder (either by placing a
compressed slug of powder into one half of the capsule, or by
filling one half of the capsule with loose powder) and the other
half of the capsule is pressed on. The advantage of inserting a
slug of compressed powder is that control of weight variation is
better. The capsules may be enterically coated before filling the
powder or after filling the powder and securing both parts
together.
[0152] Thus, for convenient and effective oral administration,
pharmaceutically effective amounts of the nanoparticles of this
invention can be tableted with one or more excipient, encased in
capsules such as gel capsules, and suspended in a liquid solution
and the like. In one embodiment, the capsules further comprise a
pharmaceutically acceptable carrier, diluent, or excipient, wherein
the excipient may comprise a solubilizer, bubbling agent, or
emulsifier. A bubbling agent is a compound that generates gas upon
contacting with liquid, such as sodium bicarbonate plus citric acid
powder or Ac-Di-Sol. The nanoparticles can be suspended in a
deionized solution or the like for parenteral administration. The
nanoparticles may be formed into a packed mass for ingestion by
conventional techniques. For instance, the nanoparticles may be
encapsulated as a "hard-filled capsule" or a "soft-elastic capsule"
using known encapsulating procedures and materials. The
encapsulating material should be highly soluble in gastric fluid so
that the particles are rapidly dispersed in the stomach after the
capsule is ingested. Each unit dose, whether capsule or tablet,
will preferably contain nanoparticles of a suitable size and
quantity that provides pharmaceutically effective amounts of the
nanoparticles. The applicable shapes and sizes of capsules may
include round, oval, oblong, tube or suppository shape with sizes
from 0.75 mm to 80 mm or larger. The volume of the capsules can be
from 0.05 cc to more than 5 cc. In one embodiment, the interior of
capsules is treated to be hydrophobic or lipophilic.
Example No. 13
In Vitro Insulin Release Study
[0153] FIG. 14 show a representative protein drug (for example,
insulin) release profile in a pH-adjusted solution for
pH-sensitivity study with an exemplary composition of CS 0.75
mg/ml, .gamma.-PGA 0.167 mg/ml, and insulin 0.083 mg/ml in
nanoparticles. In one embodiment, the exemplary composition may
include each component at a concentration range of .+-.10% as
follows: CS 0.75 mg/ml (a concentration range of 0.67 to 0.83
mg/ml), .gamma.-PGA 0.167 mg/ml (a concentration range of 0.150 to
0.184 mg/ml), and insulin 0.083 mg/ml (a concentration range of
0.075 to 0.091 mg/ml). First, solution of the insulin-loaded
nanoparticles was adjusted to pH 2.5 to simulate the gastric
environment in a DISTEK-2230A container at 37.degree. C. and 100
rpm. Samples (n=5) were taken at a pre-determined particular time
interval and the particle-free solution was obtained by
centrifuging at 22,000 rpm for 30 minutes to analyze the free or
released insulin in solution by HPLC. Until the free insulin
content in the sample solution approaches about constant of 26%
(shown in FIG. 14), the pH was adjusted to 6.6 to simulate the
entrance portion of the intestine. The net released insulin during
this particular time interval is about (from 26% to 33%) 7%. In
other words, the nanoparticles are quite stable (evidenced by
minimal measurable insulin in solution) for both the pH 2.5 and pH
6.6 regions. To further simulate the exit portion of the intestine,
the insulin-containing nanoparticle solution is adjusted to pH 7.4.
The remaining insulin (about 67%) is released from the
nanoparticles. As discussed above, the insulin in nanoparticles
would be more effective to penetrate the intestine wall in
paracellular transport mode than the free insulin because of the
nanoparticles of the present invention with chitosan at the outer
surface (preferential mucosal adhesion on the intestinal wall) and
positive charge (enhancing paracellular tight junction
transport).
Example No. 14
In Vivo Study with Insulin-Loaded Fluorescence-Labeled
Nanoparticles
[0154] In the in vivo study, rats were injected with streptozotocin
(STZ 75 mg/kg intraperitoneal) in 0.01M citrate buffer (pH 4.3) to
induce diabetes rats. The blood from the rat's tail was analyzed
with a commercially available glucometer for blood glucose. The
blood glucose level on Wistar male rats at no fasting (n=5) is
measured at 107.2.+-.8.1 mg/dL for normal rats while the blood
glucose level is at 469.7.+-.34.2 mg/dL for diabetic rats. In the
animal study, diabetic rats were fasting for 12 hours and subjected
to four different conditions: (a) oral deionized water (DI)
administration; (b) oral insulin administration at 30 U/kg; (c)
oral insulin-loaded nanoparticles administration at 30 U/kg; and
(d) subcutaneous (SC) insulin injection at 5 U/kg as positive
control. The blood glucose concentration from rat's tail was
measured over the time in the study.
[0155] FIG. 15 shows glucose change (hypoglycemic index) versus
time of the in vivo animal study (n=5). The glucose change as a
percentage of base lines for both oral DI administration and oral
insulin administration over a time interval of 8 hours appears
relatively constant within the experimental measurement error
range. It is illustrative that substantially all insulin from the
oral administration route has been decomposed in rat stomach. As
anticipated, the glucose decrease for the SC insulin injection
route appears in rat blood in the very early time interval and
starts to taper off after 3 hours in this exemplary study. The most
important observation of the study comes from the oral
administration route with insulin-loaded nanoparticles.
[0156] The blood glucose begins to decrease from the base line at
about 2 hours after administration and sustains at a lower glucose
level at more than 8 hours into study. It implies that the current
insulin-loaded nanoparticles may modulate the glucose level in
animals in a sustained or prolonged effective mode. Some aspects of
the invention provide a method of treating diabetes of a patient
comprising orally administering insulin-containing nanoparticles
with a dosage effective amount of the insulin to treat the
diabetes, wherein at least a portion of the nanoparticles comprises
a positively charged shell substrate and a negatively charged core
substrate. In one embodiment, the dosage effective amount of the
insulin to treat the diabetes comprises an insulin amount of
between about 15 units to 45 units per kilogram body weight of the
patient, preferably 20 to 40 units, and most preferably at about 25
to 35 units insulin per kilogram body weight.
[0157] Some aspects of the invention relate to a novel nanoparticle
system that is composed of a low-MW CS and .gamma.-PGA with CS
dominated on the surfaces being configured to effectively open the
tight junctions between Caco-2 cell monolayers. The surface of the
nanoparticles is characterized with a positive surface charge. In
one embodiment, the nanoparticles of the invention enables
effective intestinal delivery for bioactive agent, including
peptide, polypeptide, protein drugs, other large hydrophilic
molecules, and the like. Such polypeptide drugs can be any natural
or synthetic polypeptide that may be orally administered to a human
patient.
[0158] Exemplary drugs include, but are not limited to, insulin;
growth factors, such as epidermal growth factor (EGF), insulin-like
growth factor (IGF), transforming growth factor (TGF), nerve growth
factor (NGF), platelet-derived growth factor (PDGF), bone
morphogenic protein (BMP), fibroblast growth factor and the like;
somatostatin; somatotropin; somatropin; somatrem; calcitonin;
parathyroid hormone; colony stimulating factors (CSF); clotting
factors; tumor necrosis factors: interferons; interleukins;
gastrointestinal peptides, such as vasoactive intestinal peptide
(VIP), cholecytokinin (CCK), gastrin, secretin, and the like;
erythropoietins; growth hormone and GRF; vasopressins; octreotide;
pancreatic enzymes; dismutases such as superoxide dismutase;
thyrotropin releasing hormone (TRH); thyroid stimulating hormone;
luteinizing hormone; LHRH; GHRH; tissue plasminogen activators;
macrophage activator; chorionic gonadotropin; heparin; atrial
natriuretic peptide; hemoglobin; retroviral vectors; relaxin;
cyclosporin; oxytocin; vaccines; monoclonal antibodies; and the
like; and analogs and derivatives of these compounds.
[0159] The bioactive agent of the present invention may also be
selected from group consisting of oxytocin, vasopressin,
adrenocorticotrophic hormone, prolactin, luliberin or luteinising
hormone releasing hormone, growth hormone, growth hormone releasing
factor, somatostatin, glucagon, interferon, gastrin, tetragastrin,
pentagastrin, urogastroine, secretin, calcitonin, enkephalins,
endorphins, angiotensins, renin, bradykinin, bacitracins,
polymixins, colistins, tyrocidin, gramicidines, and synthetic
analogues, modifications and pharmacologically active fragments
thereof, monoclonal antibodies and soluble vaccines.
[0160] In another embodiment, the nanoparticles of the invention
increase the absorption of bioactive agents across the blood-brain
barrier and/or the gastrointestinal barrier. In still another
embodiment, the nanoparticles with chitosan at an outer layer and
surface positive charge serve as an enhancer in enhancing
paracellular drug (bioactive agent) transport of an administered
bioactive agent when the bioactive agent and nanoparticles are
orally administrated in a two-component system, or orally
administered substantially simultaneously.
Example No. 15
Paracellular Transport and Enhancers
[0161] Chitosan and its derivatives may function as intestinal
absorption enhancers (that is, paracellular transport enhancers).
Chitosan, when protonated at an acidic pH, is able to increase the
paracellular permeability of peptide drugs across mucosal
epithelia. Some aspects of the invention provide co-administration
of nanoparticles of the present invention and at least one
paracellular transport enhancer (in non-nanoparticle form or
nanoparticle form). In one embodiment, the nanoparticles can be
formulated by co-encapsulation of the at least one paracellular
transport enhancer and at least one bioactive agent, optionally
with other components. In one embodiment, the nanoparticles further
comprise at least one permeation enhancer. The enhancer may be
selected from the group consisting of Ca.sup.2+ chelators, EDTA
(ethylenediaminetetraacetic acid), bile salts, anionic surfactants,
medium-chain fatty acids, phosphate esters, and chitosan or
chitosan derivatives. EDTA refers to the chelating agent with the
formula
(HO.sub.2CCH.sub.2).sub.2NCH.sub.2CH.sub.2N(CH.sub.2CO.sub.2H).sub.2.
It is approved by the FDA as a preservative in packaged foods,
vitamins, and baby food. In one embodiment, the nanoparticles of
the present invention comprises a positively charged shell
substrate and a negatively charged core substrate, for example,
nanoparticles composed of .gamma.-PGA and chitosan that is
characterized with a substantially positive surface charge.
[0162] In some embodiment, the nanoparticles or particulate
complexes of the present invention and optionally the at least one
paracellular transport enhancer are encapsulated in a soft gel,
pill, enteric coated pill, enteric coated tablet, or enteric coated
capsule. The enhancers and the nanoparticles would arrive at the
tight junction about the same time for enhancing opening the tight
junction. In another embodiment, the at least one paracellular
transport enhancer is co-enclosed within the shell of the
nanoparticles of the present invention. Therefore, some broken
nanoparticles would release enhancers to assist the nanoparticles
to open the tight junctions of the epithelial layers. In an
alternate embodiment, the at least one enhancer is enclosed within
a second nanoparticle having positive surface charges, particularly
a chitosan type nanoparticle. When the drug-containing first
nanoparticles of the present invention are co-administered with the
above-identified second nanoparticles orally, the enhancers within
the second nanoparticles are released in the intestinal tract to
assist the drug-containing first nanoparticles to open and pass the
tight junction.
Example No. 16
Nanoparticles with Exenatide
[0163] Exenatide is a member of the class of drugs known as
incretin mimetics. Exenatide and pramlintide belong to non-insulin
injectables for treatment of diabetes. Exenatide has a molecular
formula of C.sub.184H.sub.282N.sub.50O.sub.60S with a molecular
mass of about 4186.6 g/mol and an CAS no. 141732-76-5. Exenatide is
suitable to be incorporated in a core portion of chitosan-shelled
nanoparticles, wherein the core portion may include positively
charged chitosan and negatively charged core substrate, such as
.gamma.-PGA or .alpha.-PGA, optionally with additional TPP and
MgSO.sub.4 in the core portion. In preparation, nanoparticles were
obtained upon addition of a mixture of .gamma.-PGA plus exenatide
aqueous solution (pH 7.4, 2 ml), using a pipette (0.5-5 ml,
PLASTIBRAND.RTM., BrandTech Scientific Inc., Germany), into a
low-MW CS aqueous solution (pH 6.0, 10 ml) at concentrations higher
than 0.10% by w/v under magnetic stirring at room temperature to
ensure positive surface charge. Nanoparticles were collected by
ultracentrifugation at 38,000 rpm for 1 hour. Exenatide is wholly
or substantially totally encapsulated in the core portion of the
nanoparticles. Supernatants were discarded and nanoparticles were
resuspended in deionized water as the solution products. In one
embodiment, it may further be encapsulated in capsules. In one
embodiment, the interior surface of the capsule is treated to be
lipophilic or hydrophobic. In another embodiment, the exterior
surface of the capsule is enteric-coated. In a preferred
embodiment, the nanoparticles are further freeze-dried, optionally
being mixed with trehalose or with hexan-1,2,3,4,5,6-hexyl in a
freeze-drying process.
[0164] Glucagon-like peptide-1 (GLP-1) is derived from the
transcription product of the proglucagon gene. The major source of
GLP-1 in the body is the intestinal L cell that secretes GLP-1 as a
gut hormone. The biologically active forms of GLP-1 are
GLP-1-(7-37) and GLP-1-(7-36)NH2. GLP-1 secretion by L cells is
dependent on the presence of nutrients in the lumen of the small
intestine. The secretagogues (agents that cause or stimulate
secretion) of this hormone include major nutrients like
carbohydrate, protein and lipid. Once in the circulation, GLP-1 has
a half-life of less than 2 minutes, due to rapid degradation by the
enzyme dipeptidyl peptidase-4 (DPP-4). Commercial GLP-1 ELISA kits
are generally available for GLP-1 assay.
[0165] Exenatide (marketed as Byetta) is the first of a new class
of medications (incretin mimetics) approved for the treatment of
type 2 diabetes. It is manufactured and marketed by Amylin
Pharmaceuticals and Eli Lilly and Company. Exenatide is a synthetic
version of exendin-4, a hormone in the saliva of the Gila monster,
a lizard native to several Southwestern American states. It
displays properties similar to human GLP-1. Exenatide is a
39-amino-acid peptide that mimics the GLP-1 incretin, an insulin
secretagogue with glucoregulatory effects. While it may lower blood
glucose levels on its own, it can also be combined with other
medications such as pioglitazone, metformin, sulfonylureas, and/or
insulin (not FDA approved yet) to improve glucose control. The
approved use of exenatide is with either sulfonylureas, metformin
or thiazolinediones. The medication is injected subcutaneously
twice per day using a pre-filled pen device.
[0166] Typical human responses to exenatide include improvements in
the initial rapid release of endogenous insulin, suppression of
pancreatic glucagon release, delayed gastric emptying, and reduced
appetite--all of which function to lower blood glucose. Whereas
some other classes of diabetes drugs such as sulfonylureas,
thiazolinediones, and insulin are often associated with weight
gain, Byetta often is associated with significant weight loss.
Unlike sulfonylureas and meglitinides, exenatide increases insulin
synthesis and secretion in the presence of glucose only, lessening
the risk of hypoglycemia. Byetta is also being used by some
physicians to treat insulin resistance.
Example No. 17
Nanoparticles with Pramlintide
[0167] Pramlintide is a synthetic amylin analogue (marketed as
Symlin). Amylin is a natural, pancreatic islet peptide that is
normally secreted with insulin in response to meals. It has several
beneficial effects on glucose homeostasis: suppression of glucagon
secretion, delaying of gastric emptying, and the promotion of
satiety. It is currently given before meals, in a separate
subcutaneous injection but usually in conjunction with insulin.
Pramlintide has a molecular formula of
C.sub.171H.sub.269N.sub.51O.sub.53S.sub.2 with a molecular mass of
about 3951.4 g/mol and an CAS no. 151126-32-8. Pramlintide
(positively charged) is currently delivered as an acetate salt.
Pramlintide is suitable to be incorporated in a core portion of a
chitosan-shelled nanoparticles, wherein the core portion may
include positively charged chitosan and negatively charged core
substrate, such as .gamma.-PGA or .alpha.-PGA, optionally with
additional TPP and MgSO.sub.4 in the core portion. In other words,
pramlintide may replace at least a portion of positively charged
chitosan in the core portion by interacting with negatively core
substrate, such as PGA, heparin or the like. In preparation,
nanoparticles were obtained upon addition of a mixture of
.gamma.-PGA plus pramlintide aqueous solution (pH 7.4, 2 ml), using
a pipette (0.5-5 ml, PLASTIBRAND.RTM., BrandTech Scientific Inc.,
Germany), into a low-MW CS aqueous solution (pH 6.0, 10 ml) at
concentrations higher than 0.10% by w/v under magnetic stirring at
room temperature to ensure positive surface charge. Nanoparticles
were collected by ultracentrifugation at 38,000 rpm for 1 hour.
Pramlintide is wholly or substantially totally encapsulated in the
core portion of the nanoparticles. Supernatants were discarded and
nanoparticles were resuspended in deionized water as the solution
products. In one embodiment, it may further be encapsulated in
capsules. In one embodiment, the interior surface of the capsule is
treated to be lipophilic or hydrophobic. In another embodiment, the
exterior surface of the capsule is enteric-coated. In a preferred
embodiment, the nanoparticles are further freeze-dried, optionally
being mixed with trehalose or with hexan-1,2,3,4,5,6-hexyl in a
freeze-drying process.
[0168] Pramlintide is an analogue of amylin, a small peptide
hormone that is released into the bloodstream by the .beta.-cells
of the pancreas along with insulin, after a meal. Like insulin,
amylin is deficient in individuals with diabetes. By augmenting
endogenous amylin, pramlintide aids in the absorption of glucose by
slowing gastric emptying, promoting satiety via hypothalamic
receptors (different receptors than for GLP-1), and inhibiting
inappropriate secretion of glucagon, a catabolic hormone that
opposes the effects of insulin and amylin.
Example No. 18
Nanoparticles with Complexed Calcitonin
[0169] Calcitonin is a protein drug that serves therapeutically as
calcium regulators for treating osteoporosis (J. Pharm. Pharmacol.
1994; 46:547-552). Calcitonin has a molecular formula of
C.sub.145H.sub.240N.sub.44O.sub.48S.sub.2 with a molecular weight
of about 3431.9 and an isoelectric point of 8.7. The net charge for
calcitonin at pH7.4 is positive that is suitable to complex or
conjugate with negatively charged core substrate, such as
.gamma.-PGA or .alpha.-PGA. In preparation, nanoparticles were
obtained upon addition of a mixture of .gamma.-PGA plus calcitonin
aqueous solution (pH 7.4, 2 ml), using a pipette (0.5-5 ml,
PLASTIBRAND.RTM., BrandTech Scientific Inc., Germany), into a
low-MW CS aqueous solution (pH 6.0, 10 ml) at concentrations higher
than 0.10% by w/v under magnetic stirring at room temperature to
ensure positive surface charge. Nanoparticles were collected by
ultracentrifugation at 38,000 rpm for 1 hour. Calcitonin is wholly
or substantially totally encapsulated in the core portion of the
nanoparticles. Supernatants were discarded and nanoparticles were
resuspended in deionized water as the solution products, further
encapsulated in capsules or further treated with an enteric
coating.
Example No. 19
Nanoparticles with Conjugated Vancomycin
[0170] Vancomycin is a protein drug that serves therapeutically as
antibiotic against bacterial pathogens. Vancomycin has a molecular
formula of C.sub.66H.sub.75N.sub.9O.sub.24 with a molecular weight
of about 1485.7 and an isoelectric point of 5.0. The net charge for
vancomycin at pH7.4 is negative that is suitable to complex or
conjugate with a portion of negatively charged shell substrate,
such as chitosan. In preparation, nanoparticles were obtained upon
addition of a mixture of .gamma.-PGA plus vancomycin aqueous
solution (pH 7.4, 2 ml), using a pipette (0.5-5 ml,
PLASTIBRAN.RTM., BrandTech Scientific Inc., Germany), into a low-MW
CS aqueous solution (pH 6.0, 10 ml) with excess concentrations
under magnetic stirring at room temperature, wherein CS
concentration is provided sufficiently to conjugate vancomycin, to
counterbalance .gamma.-PGA, and exhibit positive surface charge for
the nanoparticles. Nanoparticles were collected by
ultracentrifugation at 38,000 rpm for 1 hour. Vancomycin is wholly
or substantially totally encapsulated in the core portion of the
nanoparticles. Supernatants were discarded and nanoparticles were
resuspended in deionized water as the solution products, further
encapsulated in capsules or further treated with an enteric coating
on capsules.
[0171] Some aspects of the invention relate to a method of
enhancing intestinal or blood brain paracellular transport of
bioactive agents configured and adapted for delivering at least one
bioactive agent in a patient comprising administering nanoparticles
composed of .gamma.-PGA and chitosan, wherein the nanoparticles are
loaded with a therapeutically effective amount or dose of the at
least one bioactive agent. The nanoparticle of the present
invention is an effective intestinal delivery system for peptide
and protein drugs and other large hydrophilic molecules. In a
further embodiment, the bioactive agent is selected from the group
consisting of proteins, peptides, nucleosides, nucleotides,
antiviral agents, antineoplastic agents, antibiotics, and
anti-inflammatory drugs. In a further embodiment, the bioactive
agent is selected from the group consisting of calcitonin,
cyclosporin, insulin, oxytocin, tyrosine, enkephalin, tyrotropin
releasing hormone (TRH), follicle stimulating hormone (FSH),
luteinizing hormone (LH), vasopressin and vasopressin analogs,
catalase, superoxide dismutase, interleukin-II (IL2), interferon,
colony stimulating factor (CSF), tumor necrosis factor (TNF) and
melanocyte-stimulating hormone. In a further embodiment, the
bioactive agent is an Alzheimer antagonist.
Example No. 20
Nanoparticles with Heparin Core Substrate
[0172] Heparin is a negatively charged drug that serves
therapeutically as anti-coagulant. Heparin is generally
administered by intravenous injection. Some aspects of the
invention relate to heparin nanoparticles for oral administration
or subcutaneous administration. In a further embodiment, heparin
serves as at least a portion of the core substrate with chitosan as
shell substrate, wherein heparin conjugate at least one bioactive
agent as disclosed herein. In preparation, nanoparticles were
obtained upon addition of heparin Leo aqueous solution (2 ml),
using a pipette (0.5-5 ml, PLASTIBRAND.RTM., BrandTech Scientific
Inc., Germany), into a low-MW CS aqueous solution (pH 6.0, 10 ml)
with excess concentrations under magnetic stirring at room
temperature. Nanoparticles were collected by ultracentrifugation at
38,000 rpm for 1 hour. Heparin is wholly or substantially totally
encapsulated in the core portion of the nanoparticles. Table 4
shows the conditions of solution preparation and the average
nanoparticle size.
TABLE-US-00005 TABLE 4 Chitosan conc. Conditions Heparin conc. @2
ml @10 ml Particle size (nm) A 200 iu/ml 0.09% 298.2 .+-. 9.3 B 100
iu/ml 0.09% 229.1 .+-. 4.5 C 50 iu/ml 0.09% 168.6 .+-. 1.7 D 25
iu/ml 0.09% 140.1 .+-. 2.3
[0173] To evaluate the pH stability of the heparin-containing
nanoparticles from Example no. 20, the nanoparticles from Condition
D in Table 4 are subjected to various pH for 2 hours (sample
size=7). Table 5 shows the average size, size distribution
(polydispersity index: PI) and zeta potential (Zeta) of the
nanoparticles at the end of 2 hours under various pH environments.
The data shows the nanoparticles are relatively stable. In one
embodiment, the nanoparticles of the present invention may include
heparin, heparin sulfate, small molecular weight heparin, and
heparin derivatives.
TABLE-US-00006 TABLE 5 pH 1.5 2.6 6.6 7.4 Deionized water @5.9 Size
(nm) 150 .+-. 9 160 .+-. 12 153 .+-. 2 154 .+-. 4 147 .+-. 5 PI
0.54 .+-. 0.03 0.50 .+-. 0.04 0.08 .+-. 0.02 0.32 .+-. 0.03 0.37
.+-. 0.02 Zeta (+) 15 .+-. 2 33 .+-. 6 15 .+-. 0.1 11 .+-. 0.2 18
.+-. 4
[0174] In a further embodiment, a growth factor such as bFGF with
pharmaceutically effective amount is added to heparin Leo aqueous
solution before the pipetting step in Example No. 20. In our
laboratory, growth factors and proteins with pharmaceutically
effective amount have been successfully conjugated with heparin to
form nanoparticles of the present invention with chitosan as the
shell substrate, wherein the growth factor is selected from the
group consisting of Vascular Endothelial Growth Factor (VEGF),
Vascular Endothelial Growth Factor 2 (VEGF2), basic Fibroblast
Growth Factor (bFGF), Vascular Endothelial Growth Factor 121
(VEGF121), Vascular Endothelial Growth Factor 165 (VEGF165),
Vascular Endothelial Growth Factor 189 (VEGF189), Vascular
Endothelial Growth Factor 206 (VEGF206), Platelet Derived Growth
Factor (PDGF), Platelet Derived Angiogenesis Factor (PDAF),
Transforming Growth Factor-.beta. (TGF-.beta.), Transforming Growth
Factor-.alpha. (TGF-.alpha.), Platelet Derived Epidermal Growth
Factor (PDEGF), Platelet Derived Wound Healing Formula (PDWHF),
epidermal growth factor, insulin-like growth factor, acidic
Fibroblast Growth Factor (aFGF), human growth factor, and
combinations thereof; and the protein is selected from the group
consisting of haemagglutinin (HBHA), Pleiotrophin, buffalo seminal
plasma proteins, and combinations thereof.
[0175] In a co-pending application, U.S. patent application Ser.
No. 10/916,170 filed Aug. 11, 2004, it is disclosed that a
biomaterial with free amino groups of lysine, hydroxylysine, or
arginine residues within biologic tissues is crosslinkable with
genipin, a crosslinker (Biomaterials 1999; 20:1759-72). It is also
disclosed that the crosslinkable biomaterial may be crosslinked
with a crosslinking agent or with light, such as ultraviolet
irradiation, wherein the crosslinkable biomaterial may be selected
from the group consisting of collagen, gelatin, elastin, chitosan,
NOCC (N, O, carboxylmethyl chitosan), fibrin glue, biological
sealant, and the like. Further, it is disclosed that a crosslinking
agent may be selected from the group consisting of genipin, its
derivatives, analog (for example, aglycon geniposidic acid),
stereoisomers and mixtures thereof. In one embodiment, the
crosslinking agent may further be selected from the group
consisting of epoxy compounds, dialdehyde starch, glutaraldehyde,
formaldehyde, dimethyl suberimidate, carbodiimides, succinimidyls,
diisocyanates, acyl azide, reuterin, ultraviolet irradiation,
dehydrothermal treatment, tris(hydroxymethyl)phosphine,
ascorbate-copper, glucose-lysine and photo-oxidizers, and the
like.
[0176] In one embodiment, it is disclosed that loading drug onto a
chitosan-containing biological material crosslinked with genipin or
other crosslinking agent may be used as biocompatible drug carriers
for drug slow-release or sustained release. Several biocompatible
plastic polymers or synthetic polymers have one or more amine group
in their chemical structures, for example poly(amides) or
poly(ester amides). The amine group may become reactive toward a
crosslinking agent, such as glutaraldehyde, genipin or epoxy
compounds of the present invention. In one embodiment, the
nanoparticles comprised of crosslinkable biomaterial is
crosslinked, for example up to about 50% degree or more of
crosslinking, preferably about 1 to about 20% degree of
crosslinking of the crosslinkable components of the biomaterial,
enabling sustained biodegradation of the biomaterial and/or
sustained drug release.
[0177] By modifying the chitosan structure to alter its charge
characteristics, such as grafting the chitosan with methyl,
N-trimethyl, alkyl (for example, ethyl, propyl, butyl, isobutyl,
etc.), polyethylene glycol (PEG), or heparin (including low
molecular weight heparin, regular molecular weight heparin, and
genetically modified heparin), the surface charge density (zeta
potential) of the CS-.gamma. PGA nanoparticles may become more pH
resistant or hydrophilic. In one embodiment, the chitosan is
grafted with polyacrylic acid.
[0178] By way of illustration, trimethyl chitosan chloride might be
used in formulating the CS-.gamma. PGA nanoparticles for
maintaining its spherical biostability at a pH lower than pH 2.5,
preferably at a pH as low as 1.0. Some aspects of the invention
provide a drug-loaded chitosan-containing biological material
crosslinked with genipin or other crosslinking agent as a
biocompatible drug carrier for enhancing biostability at a pH lower
than pH 2.5, preferably within at a pH as low as 1.0.
[0179] Freeze-Dried Nanoparticles
[0180] A pharmaceutical composition of nanoparticles of the present
invention may comprise a first component of at least one bioactive
agent, a second component of chitosan (including regular molecular
weight and low molecular weight chitosan), and a third component
that is negatively charged. In one embodiment, the second component
dominates on a surface of the nanoparticle. In another embodiment,
the chitosan is N-trimethyl chitosan. In still another embodiment,
the low molecular weight chitosan has a molecular weight lower than
that of a regular molecular weight chitosan. The nanoparticles may
further comprise tripolyphosphate and magnesium sulfate. For
example, a first solution of (2 ml 0.1% .gamma.-PGA aqueous
solution @pH 7.4+0.05% Insulin+0.1% Tripolyphosphate (TPP)+0.2%
MgSO4) is added to a base solution (10 ml 0.12% chitosan aqueous
solution @pH 6.0) as illustrated in Example no. 4 under magnetic
stirring at room temperature. Nanoparticles were collected by
ultracentrifugation at 38,000 rpm for 1 hour. The bioactive agent,
the third component, tripolyphosphate and magnesium sulfate are
wholly or substantially totally encapsulated in the core portion of
the nanoparticles. Supernatants were discarded and nanoparticles
were resuspended in deionized water for freeze-drying preparation.
Other operating conditions or other bioactive agent (such as
protein, peptide, siRNA, growth factor, the one defined and
disclosed herein, and the like) may also apply.
[0181] Several conventional coating compounds that form a
protective layer on particles are used to physically coat or mix
with the nanoparticles before a freeze-drying process. The coating
compounds may include trehalose, mannitol, glycerol, and the like.
Trehalose, also known as mycose, is an alpha-linked (disaccharide)
sugar found extensively but not abundantly in nature. It can be
synthesized by fungi, plants and invertebrate animals. It is
implicated in anhydrobiosis--the ability of plants and animals to
withstand prolonged periods of desiccation. The sugar is thought to
form a gel phase as cells dehydrate, which prevents disruption of
internal cell organelles by effectively splinting them in position.
Rehydration then allows normal cellular activity to resume without
the major, generally lethal damage, which would normally follow a
dehydration/rehydration cycle. Trehalose has the added advantage of
being an antioxidant.
[0182] Trehaloze has a chemical formula as
C.sub.12H.sub.22O.sub.11.2H.sub.2O. It is listed as CAS no. 99-20-7
and PubChem 7427. The molecular structure for trehalose is shown
below.
##STR00005##
[0183] Trehalose was first isolated from ergot of rye. Trehalose is
a non-reducing sugar formed from two glucose units joined by a 1-1
alpha bond giving it the name of
.alpha.-D-glucopyranosyl-(1.fwdarw.1)-.alpha.-D-glucopyranoside.
The bonding makes trehalose very resistant to acid hydrolysis, and
therefore stable in solution at high temperatures even under acidic
conditions. The bonding also keeps non-reducing sugars in
closed-ring form, such that the aldehyde or ketone end-groups do
not bind to the lysine or arginine residues of proteins (a process
called glycation). Trehalose has about 45% the sweetness of
sucrose. Trehalose is less soluble than sucrose, except at high
temperatures (>80.degree. C.). Trehalose forms a rhomboid
crystal as the dihydrate, and has 90% of the calorific content of
sucrose in that form. Anhydrous forms of trehalose readily regain
moisture to form the dihydrate. Trehalose has also been used in at
least one biopharmaceutical formulation, the monoclonal antibody
trastuzumab, marketed as Herceptin. It has a solubility of 68.9
g/100 g H.sub.2O at 20.degree. C.
[0184] Mannitol or hexan-1,2,3,4,5,6-hexyl
(C.sub.6H.sub.8(OH).sub.6) is an osmotic diuretic agent and a weak
renal vasodilator. Chemically, mannitol is a sugar alcohol, or a
polyol; it is similar to xylitol or sorbitol. However, mannitol has
a tendency to lose a hydrogen ion in aqueous solutions, which
causes the solution to become acidic. For this, it is not uncommon
to add a substance to adjust its pH, such as sodium bicarbonate.
Mannitol has a chemical formula as C.sub.6H.sub.14O.sub.6. It is
listed as CAS no. 69-65-8 and PubChem 453. The molecular structure
for mannitol is shown below.
##STR00006##
[0185] Glycerol is a chemical compound with the formula
HOCH.sub.2CH(OH)CH.sub.2OH. This colorless, odorless, viscous
liquid is widely used in pharmaceutical formulations. Also commonly
called glycerin or glycerine, it is a sugar alcohol and fittingly
is sweet-tasting and of low toxicity. Glycerol has three
hydrophilic alcoholic hydroxyl groups that are responsible for its
solubility in water and its hygroscopic nature. Glycerol has a
chemical formula as C.sub.3H.sub.5(OH).sub.3. It is listed as CAS
no. 56-81-5. The molecular structure for glycerol is shown
below.
##STR00007##
Example No. 21
Freeze-Drying Process for Nanoparticles
[0186] Nanoparticles (at 2.5% concentration) were mixed with
solution from four types of liquid at a 1:1 volume ratio for about
30 minutes until fully dispersed. The mixed particle-liquid was
then freeze-dried under a lyophilization condition, for example, at
about -80.degree. C. and <25 mmHg pressure for about 6 hours.
The parameters in a selected lyophilization condition may vary
slightly from the aforementioned numbers. The four types of liquid
used in the experiment include: (A) DI water; (B) trehalose; (C)
mannitol; and (D) glycerol, whereas the concentration of the liquid
(A) to liquid (C) in the solution was set at 2.5%, 5% and/or 10%.
After a freeze-drying process, the mixed particle-liquid was
rehydrated with DI water at a 1:5 volume ratio to assess the
integrity of nanoparticles in each type of liquid. The results are
shown in Table 6. By comparing the particle size, polydispersity
index and zeta-potential data, only the nanoparticles from the
freeze-dried particle-trehalose runs (at 2.5%, 5%, and 10%
concentration level) show comparable properties as compared to
those of the before-lyophilization nanoparticles. Under the same
data analysis, the nanoparticles from the freeze-dried
particle-mannitol runs (at 2.5%, and 5% concentration level) show
somewhat comparable properties as compared to those of the
before-lyophilization nanoparticles.
TABLE-US-00007 TABLE 6 Properties of nanoparticles before and after
an exemplary freeze-drying process. A: DI Water B: Trehalose C:
Mannitol D: Glycerol A: DI water + NPs B: Trehalose + NPs C:
Mannitol + NPs D: Glycerol + NPs (volume 1:1), (volume 1:1),
(volume 1:1), (volume 1:1), NPs solution freeze-dried freeze-dried
freeze-dried feeze-dried Conc. 2.50% Conc. Conc. 2.50% 5.00% 10.00%
Conc. 2.50% 5.00% Conc. 2.50% 5.00% 10.00% Size 266 Size (nm)
9229.1 Size 302.4 316.7 318.9 Size (nm) 420.1 487.5 Size (nm)
6449.1 7790.3 1310.5 (nm) (nm) Kcps 352.2 Kcps 465.3 Kcps 363.7
327.7 352.2 Kcps 305.4 303.7 Kcps 796.1 356.1 493.3 PI 0.291 PI 1
PI 0.361 0.311 0.266 PI 0.467 0.651 PI 1 1 1 Zeta 25.3 Zeta Zeta
25.6 24.6 24.7 Zeta 24.4 25.3 Zeta Poten- Potential Poten-
Potential Potential tial tial
[0187] FIG. 16 shows an illustrative mechanism of nanoparticles
released from the enteric-coated capsules. FIG. 16(A) shows the
phase of nanoparticles in the gastric cavity, wherein the
freeze-dried nanoparticles 82 are encapsulated within an initial
enteric coating or coated capsule 81. FIG. 16(B) shows a schematic
of the nanoparticles during the phase of entering small intestine,
wherein the enteric coat and its associated capsule starts to
dissolve 83 and a portion of nanoparticles 82 is released from the
capsule and contacts fluid. FIG. 16(C) shows the phase of
nanoparticles in the intestinal tract, wherein the nanoparticles
revert to a wet state having chitosan at its surface. In an
alternate embodiment, nanoparticles may be released from
alginate-calcium coating. In preparation, nanoparticles are first
suspended in a solution that contains calcium chloride, wherein the
calcium ions are positively charged. With a pipette, alginate with
negatively charged carboxyl groups is slowly added to the calcium
chloride solution. Under gentle stirring, the alginate-calcium
starts to conjugate, gel, and coat on the nanoparticle surface. In
simulated oral administration of the alginate-calcium coated
nanoparticles, nanoparticles start to separate from the coating
when they enter the small intestines.
Example No. 22
Freeze-Dried Nanoparticles in Animal Evaluation
[0188] In the in vivo study, rats as prepared and conditioned
according to Example no. 14 were used in this evaluation. In the
animal evaluation study, diabetic rats were fasting for 12 hours
and subjected to three different conditions: (a) oral deionized
water (DI) administration as negative control; (b) oral
insulin-loaded lyophilized nanoparticles administration, whereas
the nanoparticles have an insulin loading content of at least 4.4%
and an insulin loading efficiency of at least 48.6% and are loaded
in a capsule with surface enteric coating; and (c) subcutaneous
(SC) insulin injection at 5 U/kg as positive control. The blood
glucose concentration from rat's tail was measured over the time in
the study.
[0189] FIG. 19 shows glucose change (hypoglycemic index) versus
time of the in vivo animal study (n=5). The glucose change as a
percentage of base lines for oral DI administration (control) over
a time interval of 10 hours appears relatively constant within the
experimental measurement error range. As anticipated, the glucose
decrease for the SC insulin injection route appears in rat blood in
the very early time interval and starts to taper off after 2 hours
in this exemplary study and ends at about 6 hours. The glucose
decrease for the orally administered insulin-loaded NPs continued
until the end of sampling time at 10 hours.
[0190] The blood glucose begins to decrease from the base line at
about 3 hours after administration and sustains at a lower glucose
level at more than 10 hours into study. It implies that the current
insulin-loaded nanoparticles may modulate the glucose level in
animals in a sustained or prolonged effective mode. Some aspects of
the invention provide a method of treating diabetes of a patient
comprising orally administering insulin-containing nanoparticles
with a dosage effective amount of the insulin to treat the
diabetes, wherein at least a portion of the nanoparticles comprises
a positively charged shell substrate and a negatively charged core
substrate. In one embodiment, the dosage effective amount of the
insulin to treat the diabetes comprises an insulin amount of
between about 15 units to 45 units per kilogram body weight of the
patient, preferably 20 to 40 units, and most preferably at about 25
to 35 units insulin per kilogram body weight. In one embodiment,
the lyophilized nanoparticles may be fed as is to an animal without
being loaded in an enterically coated capsule.
[0191] It is known that Zn (zinc) is usually added in the
biosynthesis and storage of insulin. Some aspects of the invention
relate to a nanoparticle characterized by enhancing intestinal or
blood brain paracellular transport, the nanoparticle comprising a
first component of at least one bioactive agent, a second component
of low molecular weight chitosan, and a third component that is
negatively charged, wherein a stabilizer is added to complex the at
least one bioactive agent to the negatively charged third
component. In one embodiment, the stabilizer is zinc or
calcium.
Example No. 23
Nanoparticles with Enhanced Insulin Loading
[0192] Some aspects of the invention relate to a novel nanoparticle
comprising a shell substrate of chitosan and a core substrate
consisting of at least one bioactive agent, MgSO.sub.4, TPP, and a
negatively charged substrate that is neutralized with chitosan in
the core. FIG. 10 shows insulin-loaded nanoparticles with a core
composition comprised of .gamma.-PGA, MgSO.sub.4, sodium
tripolyphosphate (TPP), and insulin. Nanoparticles were obtained
upon addition of core component, using a pipette (0.5-5 ml,
PLASTIBRAND.RTM., BrandTech Scientific Inc., Germany), into a CS
aqueous solution (pH 6.0, 10 ml) at certain concentrations under
magnetic stirring at room temperature. Nanoparticles were collected
by ultracentrifugation at 38,000 rpm for 1 hour. Supernatants were
discarded and nanoparticles were resuspended in deionized water for
further studies. In one embodiment, nanoparticles are encapsulated
in a gelcap or are lyophilized before being loaded in a gelcap or
in a tablet. The sodium tripolyphosphate has a chemical formula of
Na.sub.5P.sub.3O.sub.10 as shown below:
##STR00008##
[0193] In the example, the core composition may be varied and
evaluated with a preferred composition of 2 ml .gamma.-PGA aqueous
solution at pH 7.4 plus insulin, MgSO.sub.4 and TPP, resulting in a
ratio of CS:.gamma.-PGA:TPP:MgSO4:insulin=6.0:1.0:1.0:2.0:0.05.
Thus, the nanoparticles show characteristics with chitosan shell
and a core composition consisted of .gamma.-PGA, MgSO.sub.4, TPP,
and insulin and have an average loading efficiency of 72.8% insulin
and an average loading content of 21.6% insulin.
[0194] In the enhanced drug loading of the present example, there
provides two or more distinct ionic crosslink mechanisms. In one
embodiment, the nanoparticles of the present invention may have a
structure or matrix of interpenetrated ionic-crosslinks (that is,
elongate ionic-crosslink chains) including a first ionic-crosslink
chain of NH.sub.3.sup.+ of CS with COO.sup.- of .gamma.-PGA, a
second ionic-crosslink chain of NH.sub.3.sup.+ of CS with
SO.sub.4.sup.2- of MgSO.sub.4, a third ionic-crosslink chain of
Mg.sup.2+ of MgSO.sub.4 with COO.sup.- of .gamma.-PGA, and/or a
fourth ionic-crosslink chain of Na.sub.3P.sub.3O.sub.10.sup.2- of
TPP with NH.sub.3.sup.+ of CS or Mg.sup.2+ of MgSO.sub.4.
[0195] Some aspects of the invention relate to a nanoparticle
composition for oral administration with the insulin loading
efficiency and content at higher than 45% and 14% (preferably up to
about 73% and 22%), respectively. The prepared nanoparticles (NPs)
are stable in the range of pH 2.0 to 7.1. This broad range is to
maintain the chitosan-shelled nanoparticle and/or chitosan-shelled
nanoparticulate fragments transiently stable in most of the
intestine region (including duodenum, jejunum, and ileum) for
enhanced membrane adsorption and paracellular permeability of
active ingredient (for example, insulin, exenatide or pramlintide).
Some aspects of the invention provide a chitosan-shelled
nanoparticle with a core composition comprised of .gamma.-PGA,
MgSO.sub.4, TPP, and at least one bioactive agent, such as insulin,
exenatide or pramlintide for treatment of diabetes. In an alternate
embodiment, some aspects of the invention provide a
chitosan-shelled nanoparticle with a core composition consisted of
.gamma.-PGA, MgSO.sub.4, TPP, and at least one bioactive agent. In
one embodiment, negatively charged .gamma.-PGA may conveniently be
substituted by another negatively charge substrate, such as
heparin. In an experiment following the experimental conditions of
Example no. 23 by substituting insulin with exenatide,
chitosan-shelled nanoparticles with a core composition comprised of
.gamma.-PGA, MgSO.sub.4, TPP, and exenatide have been prepared that
exhibit similar physical and mechanical properties as compared to
the ones with insulin.
[0196] FIG. 21 shows an in vivo subcutaneous study using insulin
injectables and insulin-containing nanoparticles. The
insulin-containing nanoparticles exhibit different pharmacodynamics
and/or pharmacokinetics in a sustained releasing manner. Some
aspects of the invention relate to a pharmaceutical composition of
nanoparticles for subcutaneous or blood vessel administration in a
patient, the nanoparticles comprising a shell portion that is
dominated by positively charged chitosan, a core portion that
contains negatively charged substrate, wherein the negatively
charged substrate is at least partially neutralized with a portion
of the positively charged chitosan in the core portion, and at
least one bioactive agent loaded within the nanoparticles.
[0197] In one embodiment, the delivery route is via nasal
instillation, buccal absorption, sublingual or oral absorption. In
another embodiment, the delivery route is via endocytosis of
chitosan-shelled nanoparticles or chitosan-shelled nanoparticulate
fragments by the cells in a transcellular mode. Chitosan
nanoparticles promoted cellular uptake of its cargo via
endocytosis, a phenomenon not seen when chitosan was presented as a
soluble solution (Pharmaceutical Research 2003; 20:1812-1819). In
our lab, we have enabled endocytosis data with chitosan-shelled
bioactive nanoparticles or fragments. Endocytosis is a process
where cells absorb material (molecules such as proteins) from the
outside by engulfing it with their cell membrane. It is used by all
cells of the body because most substances important to them are
large polar molecules, and thus cannot pass through the hydrophobic
plasma membrane or cell membrane. The bioactive nanoparticle
fragments herein are generally in the range of about 10 to 150 nm,
preferably in the range of about 20 to 100 nm, and most preferably
in the range of about 20 to 50 nm.
[0198] Some aspects of the invention relate to a method of
delivering a bioactive agent to blood circulation in a patient,
comprising: (a) providing nanoparticles according to a preferred
embodiment of the pharmaceutical composition of the present
invention, wherein the nanoparticles are formed via a simple and
mild ionic-gelation method; (b) administering the nanoparticles
orally toward the intestine of the patient via stomach; (c) urging
the nanoparticles to be absorbed onto a surface of an epithelial
membrane of the intestine via muco-adhesive chitosan-shelled
nanoparticles; (d) permeating bioactive agent to pass through an
epithelial barrier of the intestine; and (e) releasing the
bioactive agent into the blood circulation. In one embodiment, the
bioactive agent is selected from the group consisting of exenatide,
pramlintide, insulin, insulin analog, and combinations thereof. In
another embodiment, the bioactive agent permeates through the tight
junctions of the epithelial membrane when chitosan-shelled
nanoparticles break up and release the bioactive agent at vicinity
of the tight junctions.
[0199] Some aspects of the invention relate to a method for
inducing a redistribution of tight junction's ZO-1 protein, leading
to translocation of the ZO-1 protein to cytoskeleton that
accompanies increased paracellular transport in a patient, the
method comprising administering into the patient bioactive
nanoparticles with a dosage effective to induce the redistribution,
wherein the bioactive nanoparticles comprise a shell substrate of
chitosan and a core substrate that comprises poly(glutamic acid)
and the bioactive agent that is selected from the group consisting
of exenatide, pramlintide, insulin, insulin analog, and
combinations thereof.
[0200] Blood-Brain Barrier and Tight Junctions
[0201] The blood-brain barrier (BBB) is a membrane structure in the
central nervous system (CNS) that restricts the passage of various
chemical substances and microscopic objects (e.g. bacteria) between
the bloodstream and the neural tissue itself, while still allowing
the passage of substances essential to metabolic function. This
"barrier" results from the selectivity of the tight junctions
between endothelial cells in CNS vessels that restricts the passage
of solutes. At the interface between blood and brain, endothelial
cells and associated astrocytes are joined together by structures
called tight junctions. The tight junction is composed of smaller
subunits, frequently dimers that are transmembrane proteins such as
occludin, claudins, junctional adhesion molecule (JAM), ESAM and
others. Each of these transmembrane proteins is anchored into the
endothelial cells by another protein complex that includes ZO-1 and
associated proteins. The blood-brain barrier is composed of
high-density cells restricting passage of substances from the
bloodstream much more than endothelial cells in capillaries
elsewhere in the body.
[0202] Some diseases associated with the blood-brain barrier may
include Meningitis, which is inflammation of the membranes that
surround the brain and spinal cord (these membranes are also known
as meninges). Meningitis is most commonly caused by infections with
various pathogens, examples of which are Staphylococcus aureus and
Haemophilus influenza. When the meninges are inflamed, the
blood-brain barrier may be disrupted. This disruption may increase
the penetration of various substances (including antibiotics) into
the brain. Some aspects of the invention relate to a method for
delivering therapeutic nanoparticles of the present invention
incorporating meningitis antagonist or anti-inflammatory drugs as a
bioactive agent to the tight junction of a brain-blood barrier site
for treatment of meningitis.
[0203] Another disease associated with brain-blood barrier may be
Epilepsy, which is a common neurological disease characterized by
frequent and often untreatable seizures. Several clinical and
experimental data have implicated failure of blood-brain barrier
function in triggering chronic or acute seizures. These findings
have shown that acute seizures are a predictable consequence of
disruption of the BBB by either artificial or inflammatory
mechanisms. In addition, expression of drug resistance molecules
and transporters at the BBB are a significant mechanism of
resistance to commonly used anti-epileptic drugs. Some aspects of
the invention relate to a method for delivering therapeutic
nanoparticles of the present invention incorporating anti-epileptic
drugs or anti-inflammatory drugs/medicine as a bioactive agent to
the tight junction of a brain-blood barrier site for treatment of
epilepsy.
[0204] Another disease associated with brain-blood barrier is
Multiple Sclerosis (MS), which is considered an auto-immune
disorder in which the immune system attacks the myelin protecting
the nerves in the central nervous system. Normally, a person's
nervous system would be inaccessible for the white blood cells due
to the blood-brain barrier. However, it has been shown using MRI
(Magnetic Resonance Imaging) that, when a person is undergoing an
MS "attack," the blood-brain barrier has broken down in a section
of the brain or spinal cord, allowing white blood cells called T
lymphocytes to cross over and destroy the myelin. It has been
suggested that, rather than being a disease of the immune system,
MS is a disease of the blood-brain barrier. It is believed that
oxidative stress plays an important role into the breakdown of the
barrier; anti-oxidants such as lipoic acid may be able to stabilize
a weakening blood-brain barrier. Some aspects of the invention
relate to a method for delivering therapeutic nanoparticles of the
present invention incorporating anti-oxidants or anti-inflammatory
medicine as a bioactive agent to the tight junction of a
brain-blood barrier site for treatment of multiple sclerosis.
[0205] One disease associated with brain-blood barrier is
Neuromyelitis optica, also known as Devic's disease, which is
similar to and often confused with multiple sclerosis. Patients
with neuromyelitis optica have high levels of antibodies against a
protein called aquaporin-4. Some aspects of the invention relate to
a method for delivering therapeutic nanoparticles of the present
invention incorporating anti-neuromyelitis optica drugs or
anti-inflammatory medicine as a bioactive agent to the tight
junction of a brain-blood barrier site for treatment of Devic's
disease.
[0206] One disease associated with brain-blood barrier is
Late-stage neurological trypanosomiasis, or sleeping sickness,
which is a condition in which trypanosoma protozoa are found in
brain tissue. It is not yet known how the parasites infect the
brain from the blood, but it is suspected that they cross through
the choroid plexus, a circumventricular organ. Some aspects of the
invention relate to a method for delivering therapeutic
nanoparticles of the present invention incorporating
anti-neurological trypanosomiasis drugs or anti-inflammatory
medicine as a bioactive agent to the tight junction of a
brain-blood barrier site for treatment of Late-stage neurological
trypanosomiasis.
[0207] One disease associated with brain-blood barrier is
Progressive multifocal leukoencephalopathy (PML), which is a
demyelinating disease of the central nervous system caused by
reactivation of a latent papovavirus (the JC polyomavirus)
infection, that can cross the BBB. Some aspects of the invention
relate to a method for delivering therapeutic nanoparticles of the
present invention incorporating anti-virus (such as papovarus)
drugs as a bioactive agent to the tight junction of a brain-blood
barrier site for treatment of PML.
[0208] One disease associated with brain-blood barrier is HIV
Encephalitis. It is believed that HIV can cross the blood-brain
barrier inside circulating monocytes in the bloodstream ("Trojan
horse theory"). Once inside, these monocytes become activated and
are transformed into macrophages. Activated monocytes release
virions into the brain tissue proximate to brain microvessels.
These viral particles likely attract the attention of sentinel
brain microglia and initiate an inflammatory cascade that may cause
tissue damage to the BBB. This inflammation is HIV encephalitis
(HIVE). Instances of HIVE probably occur throughout the course of
AIDS and is a precursor for HIV-associated dementia (HAD). Some
aspects of the invention relate to a method for delivering
therapeutic nanoparticles of the present invention incorporating
anti-HIV drugs or anti-inflammatory medicine as a bioactive agent
to the tight junction of a brain-blood barrier site for treatment
of HIV.
[0209] Among all diseases associated with blood-brain barrier, the
most critical is Alzheimer's Disease (AD). New evidence indicates
that disruption of the blood-brain barrier in AD patients allows
blood plasma containing amyloid beta (A.beta.) to enter the brain
where the A.beta. adheres preferentially to the surface of
astrocytes. These findings have led to the hypotheses that (i)
breakdown of the blood-brain barrier allows access of
neuron-binding autoantibodies and soluble exogenous A.beta.42 to
brain neurons and (ii) binding of these autoantibodies to neurons
triggers and/or facilitates the internalization and accumulation of
cell surface-bound A.beta.42 in vulnerable neurons through their
natural tendency to clear surface-bound autoantibodies via
endocytosis. Eventually the astrocyte is overwhelmed, dies,
ruptures, and disintegrates, leaving behind the insoluble A.beta.42
plaque. Thus, in some patients, Alzheimer's disease may be caused
(or more likely, aggravated) by a breakdown in the blood-brain
barrier. Some aspects of the invention relate to a method for
delivering therapeutic nanoparticles of the present invention
incorporating anti-Alzheimer's drugs (i.e., Alzheimer's antagonist)
or anti-inflammatory medicine as a bioactive agent to the tight
junction of a brain-blood barrier site for treatment of AD. In one
embodiment, the at least one bioactive agent is an antagonist for
Alzheimer's disease or is for treating Alzheimer's disease selected
from the group consisting of memantine hydrochloride, donepezil
hydrochloride, rivastigmine tartrate, galantamine hydrochloride,
and tacrine hydrochloride.
Example No. 24
Bioactive Nanoparticles Delivery Through Tight Junctions
[0210] One possible route of a drug administered by the nasal
pathway is to enter the olfactory mucosa, followed by entering the
brain tissue via cerebrospinal fluid (CSF). The mammalian nasal
cavity is lined with three types of epithelia: squamous,
respiratory and olfactory. The main part of the nasal cavity is
covered by a typical airway epithelium. CSF is secreted at the four
choroids plexi, located in the lateral and third and fourth
ventricles. CSF is an isotonic aqueous solution with the
concentrations of the major solutes practically identical to those
found in the plasma, except for K.sup.+ and Ca.sup.2+. Paracellular
passage, followed by transport through the olfactory perineural
space, that may be continuous with a subarachnoid extension that
surrounds the olfactory nerve as it penetrates the cribriform
plate, has been suggested (Arch Otolaryngology 1985; 105:180-184).
Therefore, substances may enter the brain after paracellular
passage by flushing with CSF re-entering again into the brain
extracellular space at the cribriform plate.
[0211] The olfactory system is unique because the primary neurons
of the olfactory pathway project directly to the cerebral cortex.
Consequently, the olfactory epithelium allows the influx of some
drugs into the olfactory bulb using axonal transport, and further
movement into the central nervous system. The entry of drugs into
the olfactory bulb is also possible probably by direct diffusion
into the surrounding CSF. The distribution of drugs from the nasal
membrane into the CSF appears to be controlled by a combination of
their molecular properties. For protein or peptides, the
controlling mechanism involves the tight junctions of epithelia at
the outer layer of the olfactory bulbs. The chitosan-shelled
bioactive nanoparticles or fragments of the present invention
possess the molecular properties of enhanced permeating through the
tight junctions as described above. It is generally accepted that
the nasal route circumvents the first-pass liver metabolism and
elimination associated with oral drug delivery. Some aspects of the
invention relate to a method of delivering a bioactive agent into
CSF comprising providing bioactive nanoparticles or fragments
intranasally, wherein the bioactive nanoparticles or fragments
comprise a shell substrate composed mostly of chitosan, a core
substrate that comprises the bioactive agent and a negatively
charged substrate that is at least partially neutralized with a
portion of the positively charged chitosan in the core portion.
Example No. 25
Nanoparticles with Enhanced Nasal Absorption
[0212] In contrast to oral administration, nasally administered
drugs are only transported over a very short distance, remain only
about 15 minutes in the nasal cavity and are not exposed to low pH
value and degrading enzymes. Fernandez-Urrusuno et al. reported
enhanced nasal absorption of insulin using chitosan/TPP/insulin
nanoparticles (Pharmaceutical Research 1999; 16:1576-1581), with a
mean particle size in the range of 300 nm to 400 nm and a positive
zeta potential (from +54 mV to +25 mV). The chitosan/TPP/insulin
nanoparticles have demonstrated its effect on glucose reduction in
animals by nasal instillation. The chitosan/TPP/insulin
nanoparticles as reported have shown intensified contact of insulin
with the absorptive epithelium as compared to chitosan solution. It
was suggested that the chitosan/TPP/insulin nanoparticles cross the
nasal epithelium, thus working as peptide carriers to the systemic
circulation.
[0213] In one embodiment, the nanoparticles of the present
invention may be administered to humans and other animals for
therapy by any suitable route of administrations including orally,
nasally, as by, for example, a spray, parenterally, and topically,
as by powders or drops, including orally, buccally and
sublingually. Nasal sprays can be used for transmucosal
administration. Some aspects of the invention relate to a
nanoparticle system consisting of chitosan (or chitosan derivative,
such as TMC and the like), PGA (.gamma.-PGA, .alpha.-PGA,
derivatives or salts of PGA, and the like) and at least one
bioactive agent and a method of delivering the above-mentioned
nanoparticle system or chitosan-shelled nanoparticulate fragments
via intranasal, oral, buccal or sublingual administration into the
systemic circulation. In one preferred embodiment, the systemic
circulation herein is for delivering the at least one bioactive to
brain via chitosan-shelled nanoparticles or chitosan-shelled
nanoparticulate fragments of the present invention. The bioactive
nanoparticle fragments herein are generally in the range of about
10 to 150 nm, preferably in the range of about 20 to 100 nm, and
most preferably in the range of about 20 to 50 nm.
Example No. 26
Bioactive Nanoparticles Delivery Through Systemic Blood
Circulation
[0214] The mean pH in the body fluid in intercellular spaces
between enterocytes is about 7.4. Epithelia or endothelia
constitute the structural basis of the blood-tissue barriers such
as the blood-brain, blood-nerve, blood-retina, blood aqueous and
placental barriers. Tight junctions connecting the epithelial or
endothelial cells or syncytial cell layers prevent the free
exchange of substances between the blood and the compartments
guarded by the barrier, and only limited substances are allowed to
pass through it. Many researchers have reported unique connection
between the nose and the brain and intranasal delivery of drugs to
the brain bypassing the blood-brain barrier (Eur J Pharm Sci 2000;
11:1-18; Neuroscience 2004; 127:481-496).
[0215] The nasal epithelium displays a relatively high permeability
to drugs, due to the presence of a dense blood vessel network. The
advantages of this route of administration are also a rapid
absorption and onset of the pharmacological response, avoidance of
hepatic first-pass metabolism, high systemic availability and an
easy administration route suitable for self-medication. Recently
several studies have indicated that hydrophilic or relatively large
molecules such as proteins, viruses or dextrans with a molecular
weight up to 20 KDa can be directly transported from the nasal
cavity to the CSF using neuronal anterograde and retrograde
transport, and that the transport of large molecules to the CSF is
dependent on their molecular weights. Thus a drug administered by
the nasal route may enter either the blood of the general
circulation and into CSF via choroids plexus blood-brain barrier
into the brain as described above.
[0216] The respiratory epithelium is a pseudostratified, columnar
epithelium with an abundance of secretory cells lying on a basement
membrane. It is considered the main site for drug absorption into
the systemic circulation. Intranasally delivered drugs show a rapid
rise to peak blood concentrations, due to a high permeability of
the nasal epithelia for relatively large molecules and to the
presence of an important microvasculature. As relatively large,
water-soluble peptides possess a significant nasal bioavailability,
it has been suggested that the transepithelial pathway for these
peptides is paracellular, i.e., through intercellular junctional
zones (Eur J Pharm Sci 2001; 14:69-74). Absorption enhancers are
frequently used to obtain higher bioavailability of a drug with
limited nasal absorption. Some aspects of the invention relate to
bioactive nanoparticles or fragments with chitosan (an absorption
enhancer) dominated at the surface, showing a positive surface
charge.
[0217] Chitosan-based transport system was reported for overcoming
the blood-brain barrier. Several examples illustrate intravenous
administration of a mixture of chain-like chitosan to which a
peptide was bound for treating tumor diseases or other indications
in the brain. The experimental data cited in this publication
indicate that the drug absorption in the neuronal cells can be
proven by studies in mice that had the transport system containing
chain-like chitosan. Some aspects of the present invention provide
a method of delivering a bioactive agent to CSF comprising
providing bioactive nanoparticles or fragments intranasally,
wherein the bioactive nanoparticles or fragments comprise a shell
substrate of mostly chitosan, a core substrate that comprises PGA
and the bioactive agent. In one embodiment, the bioactive
nanoparticles or fragments enters a blood vessel between the nasal
cavity and the CSF with a relatively short systemic circulation, as
compared to a long systemic circulation via conventional
intravenous injection.
[0218] In one embodiment of the present invention, the bioactive
agent is associated with or entrapped within micelles before being
loaded into the nanoparticle structure. In another embodiment, the
bioactive agent is lipophilic or hydrophobic (i.e.,
non-hydrophilic), such as Omega-3, Omega-6, Omega-9, shark liver
oil, and the like. After being associated with micelles (via a
physical, chemical, or a biochemical means), the lipophilic or
hydrophobic bioactive agent becomes encapsulatable through the
affinity of micelles toward the inactive ingredients of chitosan
and a negative charged substrate of the nanoparticles.
Lipophilicity refers to the ability of a chemical compound to
dissolve in fats, oils, lipids, and non-polar solvents such as
hexane or toluene. These non-polar solvents are themselves
lipophilic--the axiom that like dissolves like generally holds
true. Thus, lipophilic substances tend to dissolve in other
lipophilic substances, while hydrophilic (water-loving) substances
tend to dissolve in water and other hydrophilic substances.
[0219] Lipophilicity, hydrophobicity, and non-polarity are often
used interchangeably. However, the terms "lipophilic" and
"hydrophobic" are not synonymous. Lipophilic substances interact
within themselves and with other substances through the London
dispersion force. They have little to no capacity to form hydrogen
bonds. When a molecule of a lipophilic substance is enveloped by
water, surrounding water molecules enter into an `ice-like`
structure over the greater part of its molecular surface, the
thermodynamically unfavorable event that drives oily substances out
of water. Thus, lipophilic substances tend to be water
insoluble.
Example No. 27
Nanoparticles Having Bioactive Agents Associated with Micelles
[0220] Some aspects of the invention relate to bioactive
nanoparticles for enhancing the permeation of at least one
bioactive agent, the nanoparticles or collapsed nanoparticles (or
fragments thereof as defined herein comprising a portion of
collapsed nanoparticles with associated portion of the at least one
bioactive agent) comprising a shell portion that is dominated by
positively charged chitosan, a core portion that contains
negatively charged substrate, wherein the negatively charged
substrate is at least partially neutralized and/or reacted with a
portion of the positively charged chitosan in the core portion, and
the at least one bioactive agent loaded within the nanoparticles.
In one embodiment, the at least one bioactive agent is hydrophobic
or lipophilic, which is associated in micelles or loaded within
micelles before being encapsulated in nanoparticles. In one
embodiment, the micelles are made via an emulsion process, an
oil-in-water microemulsion process or self-emulsifying formulation.
In another embodiment, the self-emulsifying micelle system is a
mixture of oil, surfactant, co-surfactant and lipophilic or
hydrophobic drugs. In one embodiment, the resulting nanoparticle
with substantial CS-.gamma.-PGA uniform network across the shell
portion and the core portion enables more compact nanoparticle
framework and higher API loading.
Example No. 28
Micelles in Bioactive Nanoparticles
[0221] A micelle is an aggregate of surfactant molecules dispersed
in a liquid colloid. A typical micelle in aqueous solution forms an
aggregate with the hydrophilic "head" regions in contact with
surrounding solvent, sequestering the hydrophobic single tail
regions in the micelle centre (see FIG. 20). This phase is caused
by the insufficient packing issues of single tailed lipids in a
bilayer. The difficulty filling all the volume of the interior of a
bilayer, while accommodating the area per head group forced on the
molecule by the hydration of the lipid head group leads to the
formation of the micelle. This type of micelle is known as a normal
phase micelle (oil-in-water micelle). Inverse micelles have the
headgroups at the centre with the tails extending out (water-in-oil
micelle). Micelles are approximately spherical in shape. Other
phases, including shapes such as ellipsoids, cylinders, and
bilayers are also possible. The shape and size (typically a few
nanometers) of a micelle is a function of the molecular geometry of
its surfactant molecules and solution conditions such as surfactant
concentration, temperature, pH, and ionic strength. The process of
forming micelles is known as micellization and forms part of the
phase behavior of many lipids according to their polymorphism.
[0222] The ability of a soapy solution to act as a detergent has
been recognized for centuries. The existence of "colloidal ions" or
the highly mobile, spontaneously formed clusters came to be called
micelles. Individual surfactant molecules that are in the system
but are not part of a micelle are called "monomers." Lipid micelles
represent a molecular assembly in which the individual components
are thermodynamically in equilibrium with monomers of the same
species in the surrounding medium. In water, the hydrophilic
"heads" of surfactant molecules are always in contact with the
solvent, regardless of whether the surfactants exist as monomers or
as part of a micelle. However, the lipophilic "tails" of surfactant
molecules have less contact with water when they are part of a
micelle--this being the basis for the energetic drive for micelle
formation. In a micelle, the hydrophobic tails of several
surfactant molecules assemble into an oil-like core the most stable
form of which has no contact with water. By contrast, surfactant
monomers are surrounded by water molecules that create a "cage" of
molecules connected by hydrogen bonds. This water cage is similar
to a clathrate and has an ice-like crystal structure and can be
characterized according to the hydrophobic effect. The extent of
lipid solubility is determined by the unfavorable entropy
contribution due to the ordering of the water structure according
to the hydrophobic effect.
[0223] Micelles composed of ionic surfactants have an electrostatic
attraction to the ions that surround them in solution, the latter
known as counterions. Although the closest counterions partially
mask a charged micelle (by up to 90%), the effects of micelle
charge affect the structure of the surrounding solvent at
appreciable distances from the micelle. Ionic micelles influence
many properties of the mixture, including its electrical
conductivity. Adding salts to a colloid containing micelles can
decrease the strength of electrostatic interactions and lead to the
formation of larger ionic micelles. This is more accurately seen
from the point of view of an effective change in hydration of the
system.
[0224] Micelles only form when the concentration of surfactant is
greater than the critical micelle concentration (CMC), and the
temperature of the system is greater than the critical micelle
temperature, or Kraft temperature. The formation of micelles can be
understood using thermodynamics: micelles can form spontaneously
because of a balance between entropy and enthalpy. In water, the
hydrophobic effect is the driving force for micelle formation,
despite the fact that assembling surfactant molecules together
reduces their entropy. At very low concentrations of the lipid,
only monomers are present in true solution. As the concentration of
the lipid is increased, a point is reached at which the unfavorable
entropy considerations, derived from the hydrophobic end of the
molecule, become dominant. At this point, the lipid hydrocarbon
chains of a portion of the lipids must be sequestered away from the
water. Therefore, the lipid starts to form micelles. Broadly
speaking, above the CMC, the entropic penalty of assembling the
surfactant molecules is less than the entropic penalty of caging
the surfactant monomers with water molecules. Also important are
enthalpy considerations, such as the electrostatic interactions
that occur between the charged parts surfactants.
[0225] In a non-polar solvent, it is the exposure of the
hydrophilic head groups to the surrounding solvent that is
energetically unfavorable, giving rise to a water-in-oil system. In
this case, the hydrophilic groups are sequestered in the micelle
core and the hydrophobic groups extend away from the centre. These
inverse micelles are proportionally less likely to form on
increasing headgroup charge, since hydrophilic sequestration would
create highly unfavorable electrostatic interactions.
[0226] When surfactants are present above the CMC, they can act as
emulsifiers that will allow a compound that is normally insoluble
(in the solvent being used) to dissolve. This occurs because the
insoluble species can be incorporated into the micelle core, which
is itself solubilized in the bulk solvent by virtue of the head
groups' favorable interactions with solvent species. The most
common example of this phenomenon is detergents, which clean poorly
soluble lipophilic material (such as oils and waxes) that cannot be
removed by water alone. Detergents also clean by lowering the
surface tension of water, making it easier to remove material from
a surface. The emulsifying property of surfactants is also the
basis for emulsion polymerization.
[0227] Micelle formation is essential for the absorption of
fat-soluble vitamins and complicated lipids within the human body.
Bile salts formed in the liver and secreted by the gall bladder
allow micelles of fatty acids to form. This allows the absorption
of complicated lipids (e.g., lecithin) and lipid soluble vitamins
(A, D, E and K) within the micelle by the small intestine. FIG. 17
shows scheme of a micelle formed by phospholipids in an aqueous
solution, whereas FIG. 18 shows scheme of a micelle formed by
phospholipids in an organic solvent. In one embodiment, either
scheme of micelles may be feasible to be encapsulated in a
nanoparticle formulation as disclosed in the present invention.
Example No. 29
Emulsifying Process for Micelles Formation
[0228] An emulsion is a mixture of two or more immiscible
(unblendable) liquids. One liquid (the dispersed phase) is
dispersed in the other (the continuous phase). Many emulsions are
oil/water emulsions, with dietary fats being one common type of oil
encountered in everyday life. Examples of emulsions include butter
and margarine, milk and cream, and vinaigrettes; the
photo-sensitive side of photographic film, magmas and cutting fluid
for metal working. In butter and margarine, fat surrounds droplets
of water (a water-in-oil emulsion). In milk and cream, water
surrounds droplets of fat (an oil-in-water emulsion). In certain
types of magma, globules of liquid NiFe may be dispersed within a
continuous phase of liquid silicates. Emulsification is the process
by which emulsions are prepared.
[0229] Emulsions are thermodynamically unstable liquid/liquid
dispersions that are stabilized, in general, by surfactants.
Surfactants are usually added to emulsion systems, assembling in
the interface of the emulsion droplets, thus providing a protective
membrane that prevents the droplets from flocculating or coalescing
and thus enhancing the droplets formation and stability. Emulsion
dispersion is not about reactor blends for which one polymer is
polymerized from its monomer in the presence of the other polymers;
emulsion dispersion is a novel method of choice for the preparation
of homogeneous blends of thermoplastic and elastomer. In emulsion
dispersion system the preparation of well-fined polymers droplets
maybe acquired by the use of water as dispersing medium. The
surfactant molecules adsorb on the surface of emulsion by creating
a dispersion of droplets, which reduces interfacial tension and
retards particle flocculation during mixing. The molecules of
surfactant have polar and non-polar parts which act as an
intermediary to combine polar and non-polar polymers; the
intermolecular interactions between the polar and the non-polar
polymer segments resemble the macroscopic hydrocarbon-water
interface.
[0230] Emulsions tend to have a cloudy appearance, because the many
phase interfaces (the boundary between the phases is called the
interface) scatter light that passes through the emulsion.
Emulsions are unstable and thus do not form spontaneously. Energy
input through shaking, stirring, homogenizing, or spray processes
are needed to form an emulsion. Over time, emulsions tend to revert
to the stable state of the phases comprising the emulsion.
Surface-active substances (surfactants) can increase the kinetic
stability of emulsions greatly so that, once formed, the emulsion
does not change significantly over years of storage. Vinaigrette is
an example of an unstable emulsion that will quickly separate
unless shaken continuously. This phenomenon is called coalescence,
and happens when small droplets recombine to form bigger ones.
[0231] Emulsions are part of a more general class of two-phase
systems of matter called colloids. Although the terms colloid and
emulsion are sometimes used interchangeably, emulsion tends to
imply that both the dispersed and the continuous phase are liquid.
There are three types of emulsion instability: flocculation, where
the particles form clumps; creaming, where the particles
concentrate towards the surface (or bottom, depending on the
relative density of the two phases) of the mixture while staying
separated; and breaking and coalescence where the particles
coalesce and form a layer of liquid. Whether an emulsion turns into
a water-in-oil emulsion or an oil-in-water emulsion depends on the
volume fraction of both phases and on the type of emulsifier.
Generally, the Bancroft rule applies: emulsifiers and emulsifying
particles tend to promote dispersion of the phase in which they do
not dissolve very well; for example, proteins dissolve better in
water than in oil and so tend to form oil-in-water emulsions (that
is they promote the dispersion of oil droplets throughout a
continuous phase of water).
[0232] The basic color of emulsions is white. If the emulsion is
dilute, the Tyndall effect will scatter the light and distort the
color to blue; if it is concentrated, the color will be distorted
towards yellow. This phenomenon is easily observable on comparing
skimmed milk (with no or little fat) to cream (high concentration
of milk fat). Microemulsions and nanoemulsions tend to appear clear
due to the small size of the disperse phase.
[0233] An emulsifier (also known as an emulgent) is a substance
that stabilizes an emulsion, frequently a surfactant. Examples of
food emulsifiers are egg yolk (where the main emulsifying chemical
is lecithin), honey, and mustard, where a variety of chemicals in
the mucilage surrounding the seed hull act as emulsifiers; proteins
and low-molecular weight emulsifiers are common as well. In some
cases, particles can stabilize emulsions as well through a
mechanism called Pickering stabilization. Both mayonnaise and
Hollandaise sauce are oil-in-water emulsions that are stabilized
with egg yolk lecithin. Detergents are another class of surfactant,
and will physically interact with both oil and water, thus
stabilizing the interface between oil or water droplets in
suspension. This principle is exploited in soap to remove grease
for the purpose of cleaning. A wide variety of emulsifiers are used
in pharmacy to prepare emulsions such as creams and lotions. Common
examples include emulsifying wax, cetearyl alcohol, polysorbate 20,
and ceteareth 20.
[0234] Sometimes the inner phase itself can act as an emulsifier,
and the result is nanoemulsion--the inner state disperses into
nano-size droplets within the outer phase. A well-known example of
this phenomenon, the ouzo effect, happens when water is poured in a
strong alcoholic anise-based beverage, such as ouzo, pastis, arak
or raki. The anisolic compounds, which are soluble in ethanol, now
form nano-sized droplets and emulgate within the water. The color
of such diluted drink is opaque and milky.
[0235] Microemulsions are clear, stable, isotropic liquid mixtures
of oil, water and surfactant, frequently in combination with a
cosurfactant. The aqueous phase may contain salt(s) and/or other
ingredients, and the "oil" may actually be a complex mixture of
different hydrocarbons and olefins. In contrast to ordinary
emulsions, microemulsions form upon simple mixing of the components
and do not require the high shear conditions generally used in the
formation of ordinary emulsions. The two basic types of
microemulsions are direct (oil dispersed in water, o/w) and
reversed (water dispersed in oil, w/o). In ternary systems such as
microemulsions, where two immiscible phases (water and `oil`) are
present with a surfactant, the surfactant molecules may form a
monolayer at the interface between the oil and water, with the
hydrophobic tails of the surfactant molecules dissolved in the oil
phase and the hydrophilic head groups in the aqueous phase. As in
the binary systems (water/surfactant or oil/surfactant),
self-assembled structures of different types can be formed,
ranging, for example, from (inverted) spherical and cylindrical
micelles to lamellar phases and discontinuous microemulsions, which
may coexist with predominantly oil or aqueous phases.
[0236] The microemulsion region is usually characterized by
constructing ternary-phase diagrams. Three components are the basic
requirement to form a microemulsion: an oil phase, an aqueous phase
and a surfactant. If a cosurfactant is used, it may sometimes be
represented at a fixed ratio to surfactant as a single component,
and treated as a single "pseudo-component". The relative amounts of
these three components can be represented in a ternary phase
diagram. Gibbs phase diagrams can be used to show the influence of
changes in the volume fractions of the different phases on the
phase behavior of the system. The three components composing the
system are each found at an apex of the triangle, where their
corresponding volume fraction is 100%. Moving away from that corner
reduces the volume fraction of that specific component and
increases the volume fraction of one or both of the two other
components. Each point within the triangle represents a possible
composition of a mixture of the three components or
pseudo-components, which may consist (ideally, according to the
Gibbs' phase rule) of one, two or three phases. These points
combine to form regions with boundaries between them, which
represent the "phase behavior" of the system at constant
temperature and pressure.
[0237] Some aspects of the invention provide a formulation of
micelles and methods of formulating micelles, the micelles
comprising a basic structure as described above and at least one
hydrophobic or lipophilic bioactive agent enclosed within (called
`bioactive micelles`). One aspect of the invention further provides
a pharmaceutical composition of nanoparticles, the nanoparticles
comprising a shell portion that is dominated by positively charged
chitosan, a core portion that comprises one negatively charged
substrate, wherein the negatively charged substrate is at least
partially neutralized with a portion of the positively charged
chitosan in the core portion, and micelles, wherein at least one
bioactive agent is loaded within the micelles. The micelles are
less than about 100 nanometers, preferably less than about 20
nanometers, and most preferably less than about 10 nanometers.
Example No. 30
Chitosan/DNA Complexes Coated by Anionic Compound
[0238] In a non-viral carrier for gene delivery, a particle complex
(or particulate complex) system comprised of the core CS/DNA
complex and the outer coating of an anionic polymer, for example
poly(.gamma.-glutamic acid) (.gamma.-PGA) makes the particulate
complex more compact because .gamma.-PGA was entangled tightly with
the excess CS emanating from the surface of core CS/DNA complexes
(also known as test complexes). In one embodiment, the particulate
complex is in a nanoparticle or nanoparticle-like configuration. In
another embodiment, the core CS/DNA complex is manufactured via a
simple and mild ionic gelation method similar to the method as
disclosed in Example No. 4, wherein CS is a cationic
amine-containing polysaccharide and DNA is a negatively-charged
substrate. In one embodiment, the particulate complexes have a mean
particle size between about 50 and 400 nanometers.
[0239] Some aspects of the invention relate to a method of
manufacturing a bioactive agent-containing particulate complex
comprising the steps of (a) forming the core complex by mixing the
positively charged chitosan and a first negatively charged
substrate in a gelation process, including the bioactive agent; (b)
coating a second negatively charged substrate onto the surface of
the core complex to form the particulate complex suspension in
solution, and (c) separate the bioactive agent-containing
particulate complexes from the solution. In one embodiment, the
bioactive agent is DNA, whereas DNA is a negatively charged
substrate. In another embodiment, the first or second negatively
charged substrate is .gamma.-PGA, which is selected from the group
consisting of .gamma.-PGA, .alpha.-PGA, PGA derivatives, PGA-DTPA,
acetylated .gamma.-PGA and salts of PGA. In another exemplary
embodiment, the particulate complex is CS/DNA/.gamma.-PGA complex.
.gamma.-PGA can be in different conformational states, including
.alpha.-helix, f3-sheet, random coil, helix-coil transition and
enveloped aggregation. In DI water (pH 6.0), .gamma.-PGA molecules
are mainly in the form of linear random-coil conformation and show
a polyanionic characteristic, allowing themselves to entangle
tightly with the excess CS emanating from the surface of the core
complex, thus making the particulate complex more compact. In one
embodiment, the particulate complexes are treated with an enteric
polymer.
[0240] FIG. 23 shows the particle size and zeta potential of the
particulate complexes in aqueous media, where the core complexes
coated with .gamma.-PGA had a smaller particle size than their
counterpart (core complex in the absence of .gamma.-PGA coating).
It was evident that .gamma.-PGA coating significantly reduced their
surface charge. With increasing the coating of .gamma.-PGA, the
zeta potential of the particulate complexes decreased appreciably
and reached its minimum value (approximately -27 mV) at an (N/P)/C
ratio of (10/1)/12. Further increasing the amount of .gamma.-PGA
onto the core complex did not significantly alter the surface
charge of the particulate complexes, as shown in the cases of
(N/P)/C ratios of (10/1)/20 and (10/1)/40. In one embodiment as
shown in FIG. 23, the surface zeta potential of the particulate
complexes may be positive, neutral, or negative values.
[0241] With .gamma.-PGA coating on core CS/DNA complex, the extent
of complexes internalization and their transfection efficiency were
evidently enhanced. The endocytosis inhibition study indicates that
the .gamma.-glutamyl transpeptidase (GGT) present on cell membranes
was responsible for the uptake of core complexes. In one
embodiment, when entangled with CS, the free N-terminal
.gamma.-glutamyl unit of .gamma.-PGA on complexes was exposed and
thus be accommodated within the .gamma.-glutamyl binding pocket of
the membrane GGT. After internalization of the particulate complex,
as both .gamma.-PGA and DNA in the complexes carried negative
charges, the electrostatic repulsion between the two might lead to
the disintegration of the complexes. Such a structure disruption
may facilitate the intracellular release of DNA, thus augmenting
their transfection efficiency.
[0242] Some aspects of the invention relate to CS/DNA/.gamma.-PGA
particulate complexes that significantly enhance the cellular
uptake of complexes, consequently augmenting their gene expressing
level. Some aspects of the invention relate to a composition of
particulate complexes, the complexes consisting of a core portion
of positively charged chitosan, a first negatively charged
substrate, optionally a zero-charge compound and one bioactive
agent, and an outer portion of a second negatively charged
substrate wherein the particulate complexes have a mean complex
size between about 50 and 400 nanometers. In one embodiment, the
first negatively charged substrate is DNA, RNA, a small interfering
ribonucleic acid (siRNA), or the like. In one embodiment, the
bioactive agent is selected from the group consisting of hormone,
growth hormone, and human growth hormone. In one embodiment, the
core portion further comprises micelles, wherein the micelles are
oil-in-water micelles, water-in-oil micelles, or hybrid
micelles.
[0243] Although the present invention has been described with
reference to specific details of certain embodiments thereof, it is
not intended that such details should be regarded as limitations
upon the scope of the invention except as and to the extent that
they are included in the accompanying claims. Many modifications
and variations are possible in light of the above disclosure.
* * * * *