U.S. patent application number 15/181944 was filed with the patent office on 2016-11-03 for nano delivery systems for sirna.
This patent application is currently assigned to YISSUM RESEARCH DEVELOPMENT COMPANY OF THE HEBREW UNIVERSITY OF JERUSALEM LT. The applicant listed for this patent is YISSUM RESEARCH DEVELOPMENT COMPANY OF THE HEBREW UNIVERSITY OF JERUSALEM LT. Invention is credited to Orit AMSALEM, Simon BENITA, Taher NASSAR.
Application Number | 20160317460 15/181944 |
Document ID | / |
Family ID | 47192058 |
Filed Date | 2016-11-03 |
United States Patent
Application |
20160317460 |
Kind Code |
A1 |
BENITA; Simon ; et
al. |
November 3, 2016 |
NANO DELIVERY SYSTEMS FOR SIRNA
Abstract
The present invention makes use of a unique methodology of
double nano-encapsulation for protecting and controlling the
release of active agents, either hydrophobic or hydrophilic, from
stable nanoparticles of opposite characteristics. The protection of
the active agent was achieved by loading the agent to be protected,
into nanocarriers, which were subsequently encapsulated into
sub-micron nanoparticles. The sub-micron nanoparticles formation
has been successfully achieved by the use of novel nanospray
techniques.
Inventors: |
BENITA; Simon; (Tel Aviv,
IL) ; AMSALEM; Orit; (Jerusalem, IL) ; NASSAR;
Taher; (Kfar Turaan, IL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
YISSUM RESEARCH DEVELOPMENT COMPANY OF THE HEBREW UNIVERSITY OF
JERUSALEM LT |
Jerusalem |
|
IL |
|
|
Assignee: |
YISSUM RESEARCH DEVELOPMENT COMPANY
OF THE HEBREW UNIVERSITY OF JERUSALEM LT
Jerusalem
IL
|
Family ID: |
47192058 |
Appl. No.: |
15/181944 |
Filed: |
June 14, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14345154 |
Mar 14, 2014 |
9421173 |
|
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PCT/IL2012/050382 |
Sep 20, 2012 |
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15181944 |
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61537241 |
Sep 21, 2011 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 2320/52 20130101;
C07K 16/22 20130101; C12N 2810/859 20130101; A61K 9/1271 20130101;
C12N 15/1138 20130101; C07K 2317/55 20130101; A61K 31/713 20130101;
C12N 2310/14 20130101; C07K 2317/24 20130101; A61K 9/1272 20130101;
A61K 9/5169 20130101; A61K 47/6925 20170801; A61K 9/5153 20130101;
A61K 9/0019 20130101; A61K 9/5146 20130101; C12N 15/88 20130101;
A61K 9/5192 20130101 |
International
Class: |
A61K 9/51 20060101
A61K009/51; C07K 16/22 20060101 C07K016/22; C12N 15/113 20060101
C12N015/113 |
Claims
1. A polymeric nanoparticle encapsulating a plurality of
nanocarriers, at least a portion of the nanocarriers containing at
least one active agent, the nanoparticle having an averaged
diameter of between 400 and 950 nm, wherein the plurality of
nanocarriers being made of a material such that (i) where the
active agent is hydrophobic, the nanocarrier material is
hydrophobic and the nanoparticle material is hydrophilic; (ii)
where the active agent is hydrophilic, the nanocarrier material is
hydrophilic and the nanoparticle material is hydrophobic; (iii)
where the active agent is hydrophobic, the nanocarrier material is
hydrophobic and the nanoparticle material is hydrophobic; and (iv)
where the active agent is hydrophilic, the nanocarrier material is
hydrophilic, and the nanoparticle material is hydrophilic.
2. The nanoparticle of claim 1, wherein the nanoparticles and/or
the nanocarriers are each obtainable by nanospraying.
3. The nanoparticle of claim 1, wherein the nanoparticle and/or the
nanocarrier are each cross-linked.
4. The nanoparticle of claim 1, wherein the nanocarrier further
comprises a polycationic lipid being
1,2-dioleoyl-3-trimethylammonium-propane (DOTAP).
5. The nanoparticle of claim 1, wherein the average diameter of
said nanocarrier is less about 300 nm.
6. The nanoparticle of claim 1, wherein the nanoparticle and/or the
nanocarrier being each in the form selected from a nanocapsule or a
nanosphere.
7. The nanoparticle of claim 1, wherein said hydrophobic material
is selected from the group consisting of lactic acid,
poly(D,L-lactic-co-glycolic acid) (PLGA), poly(D,L-lactic acid)
(PLA), poly(.epsilon.-caprolactone),
poly(2-dimethylamino-ethylmethacrylate) homopolymer,
poly(2-dimethylamino-ethylmethacrylate)-b-poly(ethyleneglycop-.alpha.-met-
hoxy-.omega.-metacrylate copolymers, polycyanoacrylates,
polyanhydride polymers and combinations thereof.
8. The nanoparticle of claim 7, wherein said PLGA has a molecular
weight of between about 4,000 and 100,000 Da.
9. The nanoparticle of claim 1, wherein said nanoparticle material
is hydrophobic, an outer surface of said nanoparticle being
associated with at least one polyethylene glycol (PEG) moiety.
10. The nanoparticle of claim 1, wherein said hydrophilic material
is selected from the group consisting of dextran, hylauronate,
human serum albumin (HSA), being normal or cross-linked, bovine
serum albumin (BSA) being normal or cross-linked, chitosan,
shellac, collagen, gelatin, gum arabic, polyvinyl alcohol,
cyclodextrin, and combinations thereof.
11. The nanoparticle of claim 1, wherein said active agent is
selected from a vitamin, a protein, an anti-oxidant, a nucleic
acid, a short or long oligonucleotide, a siRNA and its chemical
derivatives, a peptide, a polypeptide, a lipid, a carbohydrate, a
hormone, an antibody, a monoclonal antibody, a vaccine, a
prophylactic agent, a drug, a diagnostic agent, a contrasting
agent, a nutraceutical agent, a small molecule, an electrolyte, an
immunological agent and combination thereof.
12. The nanoparticle of claim 11, wherein the nanocarrier comprises
at least one hydrophilic active agent selected from the group
consisting of exenatide, insulin, growth hormone, triptorelin
acetate, buserelin, and nafarelin.
13. The nanoparticle of claim 11, wherein the nanocarrier comprises
at least one hydrophobic active agent selected from the group
consisting of an analgesic or anti-inflammatory agent; an
enthelmintic agent; an anti-arrhythmic agent; an anti-bacterial
agent; an anti-coagulant; an anti-depressant; an antidiabetic; an
anti-epileptic; an anti-fungal agent; an anti-gout agent; an
anti-hypertensive agent; an anti-malarial agent; an anti-migraine
agent; an anti-muscarinic agent; an anti-neuroplastic agent or
immunosuppressant; an anti-protazoal agent; an anti-thyroid agent;
an alixiolytic, sedative, hypnotic or neuroleptic agent; a
beta-blocker; a cardiac inotropic agent; a corticosteroid; a
diuretic agent; an anti-Parkinsonian agent; a gastro-intestinal
agent; an histamine H1-receptor antagonist; a lipid regulating
agent; a nitrate or anti-anginal agent; a nutritional agent; an HIV
protease inhibitor; an opioid analgesic; a sex hormone; and a
stimulant agent.
14. The nanoparticle of claim 1, having an outer surface associated
with at least one targeting agent, optionally selected from the
group consisting of a monoclonal antibody; a small molecule;
hyaluronic acid or hyaluronan; tumor penetrating peptides;
epidermal growth factor (EGF); transferrin; ferritin;
Arginine-Glycine-Aspartic acid (RGD) peptide; epithelial cell
adhesion molecule (EpCAM); intercellular adhesion molecule 1
(ICAM-1); carcinoembrionic antigen (CEA); vasoactive intestinal
peptide; CA 15-3 antigen; MUC1 protein; CD20; CD33; integrins;
lymphatic targeting moieties (such as LyP-1); aptamers; and
oligosaccharides.
15. A composition comprising a plurality of nanoparticles of claim
1.
16. A process for encapsulating a plurality of nanocarriers in a
polymeric nanoparticle, the process comprising: (a) obtaining at
least one nanocarrier, said nanocarrier comprising at least one
active agent; and (b) encapsulating a plurality of nanocarriers
into said nanoparticle by nanospray drying.
17. The process of claim 16, wherein the nanocarriers are obtained
by: dissolving a hydrophobic polymer in organic solvent to form an
organic phase; contacting said organic phase with an aqueous phase,
the aqueous phase optionally comprising a surfactant, to thereby
obtain said nanocarriers; and incubating said nanocarriers with a
solution of said active agent to allow association of said active
agent with at least a portion of the surface of said
nanocarriers.
18. The process of claim 16, wherein the nanocarriers are obtained
by: dissolving a hydrophilic polymer in an aqueous solution of the
active agent to form an aqueous phase; and continuously adding an
organic phase comprising a desolvating agent to the aqueous phase
under a pH permitting the formation said nanocarriers, the active
agent being distributed within the nanocarrier.
19. The process of claim 16, further comprising lyophilizing the
nanoparticle.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This is a Continuation of application Ser. No. 14/345,154
filed Mar. 14, 2014, which in turn is a national phase of
PCT/IL2012/050382, filed Sep. 20, 2012, which claims the benefit of
U.S. Provisional Application No. 61/537,241 filed Sep. 21, 2011.
The disclosure of the prior applications is hereby incorporated by
reference herein in its entirety.
FIELD OF THE INVENTION
[0002] This invention generally relates to nano delivery
systems.
BACKGROUND
[0003] The discovery of RNA interference (RNAi) has opened up an
entirely new field of biology and medicine. The ability of RNAi to
specifically silence target genes has yielded not only a new tool
for basic research but also raised the concept of developing
medicines based on RNAi. RNAi works through the targeting of mRNA
via sequence-specific matches and results in degradation of target
mRNA or its translational inhibition, leading to the loss of
protein expression. This is pharmacologically achieved via the
introduction of small 19-21 bp dsRNA molecules called small
interfering RNA (siRNA). Since its discovery 10 years ago, siRNA
has been widely investigated in vitro for its utility in treating
various diseases, such as cancer, neurodegenerative and infectious
diseases.
[0004] A major barrier to further development of siRNA has been the
inability to effectively deliver siRNA in vivo due to the large
molecular weight (for example, 13 kDa) and polyanionic nature (e.g.
40 negative phosphate charges). Naked siRNA does not freely cross
the cell membrane. Furthermore, unmodified, naked siRNAs are
relatively unstable in blood and serum, as they are rapidly
degraded by endo- and exonucleases, meaning that they have short
half-lives in vivo. Typically, chemical modifications can be
introduced into the RNA duplex structure so as to enhance
biological stability without adversely affecting gene-silencing
activity. Alternatively, they can be formulated with a delivery
system that not only enhances cell uptake but also affords
biological stability. Several chemical modifications to the
backbone, base, or sugar of the RNA have been employed to enhance
siRNA stability and activity. However, delivery systems are still
required to facilitate siRNA access to its intracellular sites of
action.
[0005] Indeed, various delivery systems have been developed to
enhance the uptake of siRNA into the target tissues after systemic
administration. These include the use of polymers [1], lipids [2]
or nanoparticles [3,4]. Most of these vectors are cationic to
ensure efficient interaction of particles with negatively-charged
siRNA nucleotides and to facilitate their cell entry. However, the
ability of these cationic particles to deliver siRNA systemically
is often poor due to rapid uptake by reticuloendothelial (RES)
organs [5], thereby hindering the delivery of these particles to
the site of interest. To overcome this problem, polyethylene glycol
(PEG) has been used extensively in the formulation as it decreases
RES uptake of these particles. This PEGylation also permits the
accumulation of the particles in sites where defective vasculature
is present, such as tumors, owing to the "Enhanced Permeability and
Retention" phenomenon [6].
[0006] For lipid-based delivery vectors, various methods for
formulating polynucleotide-loaded PEGylated particles have been
reported to date, including post-insertion [7], reverse-phase
evaporation [8], detergent dialysis [9] and ethanol dialysis [10].
However, most of these methods, though effective, require
relatively complicated and lengthy formulation procedures with the
resulting particles suspended in an aqueous state. This has led to
long-term storage issues including aggregation and/or fusion of the
particles, hydrolysis of the lipids, and instability of siRNA
nucleotides in an aqueous environment. Moreover, these formulations
are also prone to be affected by stresses occurring during
transport, such as agitation or temperature fluctuation [11]. These
problems, along with the significantly increased effort required
for large-scale production of these particles using the existing
formulation procedures will limit the widespread adoption of
siRNA-containing lipid-based products in the clinics. Clearly,
there is a need to develop relatively simple and effective method
to formulate siRNA-loaded nanocarriers where the final product is
also suitable for ling term storage.
[0007] In the past two decades, several therapeutics based on
nanosized particles in the range of 1-1,000 nm have been
successfully introduced for the treatment of cancer, pain, and
infectious disease. Hydrophilic bio-macromolecules (such as
peptides or siRNA), usually exhibiting poor membrane permeability
and high sensitivity to environmental conditions (heat, pH,
enzymatic degradation) are considered adequate candidates for
intracellular delivery by means of nanocarriers. Such nanocarriers
can prolong the blood circulation time of these macromolecules
which suffer from short physiological half lives, followed by a
rapid clearance. However, the number of clinically relevant
nanocarriers used for such a purpose is scarce, and major
challenges still remain to be solved, especially for their
efficient delivery via the parenteral route of administration.
siRNAs represent a class of hydrophilic bio-macromolecules where
the application of appropriate nanocarriers is most needed to
exploit their full therapeutic potential.
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SUMMARY OF THE INVENTION
[0032] The present invention makes use of a unique methodology of
double nano-encapsulation for protecting and controlling the
release of active agents, either hydrophobic or hydrophilic agents,
such as siRNA (or its different chemical derivatives such as
cholesterol labeled siRNA), from stable nanoparticles of opposite
characteristics (hydrophobic or hydrophilic). The protection of the
active agent was achieved by loading the agent to be protected,
into nanocarriers, which were subsequently encapsulated into
sub-micron nanoparticles. The sub-micron nanoparticles
(nanocapsules or nanospheres) formation has been successfully
achieved by the use of novel nanospray techniques.
[0033] The delivery systems of the invention provide a platform for
systemic delivery of hydrophilic bio-macromolecules (such as siRNA)
or hydrophobic bio-macromolecules improving the drug's half-life,
biodistribution and pharmacokinetics.
[0034] Thus, the present invention also relates to a drug delivery
system comprising double-encapsulated active agents, enabling
stabilization of either hydrophilic or hydrophobic active agents,
and permitting their targeted delivery into cells.
[0035] In one of its aspects, the invention provides a nanoparticle
encapsulating a plurality of nanocarriers (one or more), at least
one of said plurality of nanocarriers containing at least one
active agent, said nanoparticle having an averaged diameter of
between 400 and 950 nm.
[0036] In some embodiments, the double-encapsulation of the active
agent to form nanoparticles according to the invention is
obtainable by nanospraying, as detailed herein. The process of
nanospraying may further comprise drying the nanoparticles obtained
by the nanospraying method. The drying may be achieved by
evaporation of the media solvents by using, for example,
lyophillization, thermal drying, reduced pressure, solvent
extraction and other techniques.
[0037] In additional embodiments, the nanoparticles are selected
from: [0038] i. where the active agent is hydrophobic, the
nanocarrier material is hydrophobic and the nanoparticle material
is hydrophilic; [0039] ii. where the active agent is hydrophilic,
the nanocarrier material is hydrophilic and the nanoparticle
material is hydrophobic; [0040] iii. where the active agent is
hydrophobic, the nanocarrier material is hydrophobic and the
nanoparticle material is hydrophobic; and [0041] iv. where the
active agent is hydrophilic, the nanocarrier material is
hydrophilic, and the nanoparticle material is hydrophilic.
[0042] In other embodiments, the active agent is siRNA. In some
embodiments, where the active agent is siRNA, the nanocarrier
further comprises a polycationic lipid. In some embodiments, the
polycationic lipid is 1,2-dioleoyl-3-trimethylammonium-propane
(DOTAP).
[0043] As may be understood by a person versed in the art, the term
"material" refers to the material(s) from which the nanoparticle or
nanocarrier is made of. Thus, the term "nanocarrier material"
refers to material(s) from which the nanocarrier is made of.
Similarly, the "nanoparticle material" is the material(s) from
which the nanoparticle is made of. The plurality of nanocarriers
may be made of different materials. The nanocarrier material(s) is,
in some embodiments, different from the nanoparticle material.
[0044] In some embodiments, the nanocarrier may be composed of a
metallic material, or may contain a metallic material in
combination with a non-metallic material. In some embodiments, the
nanocarriers are gold nanospheres.
[0045] The material hydrophobicity or hydrophilicity may be due to
the material intrinsic behaviors towards water, as further
discussed below, or may be achieved (or tuned) by modifying the
material by one or more of cross-linking said material,
derivatization of the material, charge induction to said material
(rendering it positively or negatively charged), complexing or
conjugating said material to another material and by any other
means known in the art.
[0046] Thus, in accordance with the present invention, the
selection of a material may be based on the material intrinsic
properties or based on the material's ability to undergo such
aforementioned modification to render it more or less hydrophobic
or hydrophilic.
[0047] In some embodiments, the nanoparticle material and/or the
nanocarrier material may be cross-linked in order to reduce
material hydrophilicity (decrease solubility in aqueous media).
[0048] In another aspect of the present invention, there is
provided a nanoparticle encapsulating a plurality of nanocarriers,
at least one of said plurality of nanocarriers (one or more)
containing at least one active agent, said nanoparticle being
prepared by nanospraying, as defined herein.
[0049] In some embodiments, the nanoparticles have an averaged
diameter of less than 4 micron. In other embodiments, the
nanoparticles have an averaged diameter of less than 2 micron. In
other embodiments, the nanoparticles have an averaged diameter of
less than 1 micron. In further embodiments, said nanoparticles
having averaged diameter of less than 950 nm. In some embodiments,
the formed nanoparticles have averaged diameter of between 400 and
950 nm.
[0050] In some embodiments, the nanoparticles are selected from:
[0051] i. where the active agent is hydrophobic, the nanocarrier
material is hydrophobic and the nanoparticle material is
hydrophilic; [0052] ii. where the active agent is hydrophilic, the
nanocarrier material is hydrophilic and the nanoparticle material
is hydrophobic; [0053] iii. where the active agent is hydrophobic,
the nanocarrier material is hydrophobic and the nanoparticle
material is hydrophobic; and [0054] iv. where the active agent is
hydrophilic, the nanocarrier material is hydrophilic, and the
nanoparticle material is hydrophilic.
[0055] In further embodiments, the nanoparticle material and/or the
nanocarrier material may be cross-linked in order to reduce
material hydrophilicity (decrease solubility in aqueous media).
[0056] In other embodiments, the active agent is siRNA. In some
embodiments, where the active agent is siRNA, the nanocarrier
further comprises a polycationic lipid. In some embodiments, the
polycationic lipid is 1,2-dioleoyl-3-trimethylammonium-propane
(DOTAP).
[0057] In another aspect of the invention, there is provided a
nanoparticle encapsulating a plurality of nanocarriers (one or
more), at least one of said plurality of nanocarriers containing at
least one active agent, such that:
[0058] where the active agent is hydrophobic, the nanocarrier
material is hydrophobic and the nanoparticle material is
hydrophilic; the nanoparticle material being optionally further
cross-linked to reduce their solubility in aqueous media; or
[0059] where the active agent is hydrophilic, the nanocarrier
material is hydrophilic, optionally cross-linked, and the
nanoparticle material is hydrophobic.
[0060] In some embodiments, the nanoparticles have an averaged
diameter of less than 4 micron. In other embodiments, the
nanoparticles have an averaged diameter of less than 2 micron. In
other embodiments, the nanoparticles have an averaged diameter of
less than 1 micron. In further embodiments, said nanoparticles
having averaged diameter of less than 950 nm. In still additional
embodiments, the nanoparticles have averaged diameter of between
400 and 950 nm.
[0061] In some embodiments, said nanoparticles are formed by
nanosparying-drying.
[0062] In other embodiments, the active agent is hydrophilic. In
some embodiments, where the active agent is siRNA, the hydrophilic
active agent is siRNA. In such embodiments, the nanocarrier further
comprises a polycationic lipid. In some embodiments, the
polycationic lipid is 1,2-dioleoyl-3-trimethylammonium-propane
(DOTAP).
[0063] In another aspect of the invention, there is provided a
nanoparticle encapsulating a plurality of nanocarriers, at least
one of said plurality of nanocarriers (one or more) containing at
least one active agent, wherein said nanoparticle having averaged
diameter of between 400 and 950 nm, wherein:
[0064] where the active agent is hydrophobic, the nanocarrier
material is hydrophobic and the nanoparticle material is
hydrophilic; the nanoparticle material being optionally further
cross-linked to reduce their solubility in aqueous media; or where
the active agent is hydrophilic, the nanocarrier material is
hydrophilic, optionally cross-linked, and the nanoparticle material
is hydrophobic.
[0065] In some embodiments, said nanoparticles are formed by
nanospraying.
[0066] In other embodiments, the active agent is hydrophilic. In
some embodiments, where the active agent is siRNA, the hydrophilic
active agent is siRNA. In such embodiments, the nanocarrier further
comprises a polycationic lipid. In some embodiments, the
polycationic lipid is 1,2-dioleoyl-3-trimethylammonium-propane
(DOTAP).
[0067] In another aspect of the invention, there is provided a
nanoparticle encapsulating a plurality of nanocarriers (one or
more), at least one of said plurality of nanocarriers containing at
least one active agent, wherein said nanoparticle having averaged
diameter of between 400 and 950 nm, wherein:
[0068] where the active agent is hydrophobic, the nanocarrier
material is hydrophobic and the nanoparticle material is
hydrophilic; the nanoparticle material being optionally further
cross-linked to reduce their solubility in aqueous media; or
[0069] where the active agent is hydrophilic, the nanocarrier
material is hydrophilic, optionally cross-linked, and the
nanoparticle material is hydrophobic;
[0070] wherein said nanoparticles are formed by nanospraying.
[0071] In yet another aspect, the invention provides a nanoparticle
encapsulating a plurality of nanocarriers (one or more), at least
one of said plurality of nanocarriers containing at least one
active agent, wherein said nanoparticle having averaged diameter of
between 400 and 950 nm, said nanoparticle being prepared by
nanospraying, as defined herein.
[0072] In some embodiments, the nanoparticles are selected from:
[0073] i. where the active agent is hydrophobic, the nanocarrier
material is hydrophobic and the nanoparticle material is
hydrophilic; [0074] ii. where the active agent is hydrophilic, the
nanocarrier material is hydrophilic and the nanoparticle material
is hydrophobic; [0075] iii. where the active agent is hydrophobic,
the nanocarrier material is hydrophobic and the nanoparticle
material is hydrophobic; and iv. where the active agent is
hydrophilic, the nanocarrier material is hydrophilic, and the
nanoparticle material is hydrophilic.
[0076] In further embodiments, the nanoparticle material and/or the
nanocarrier material may be cross-linked in order to reduce
material hydrophilicity (decrease solubility in aqueous media).
[0077] In other embodiments, the active agent is siRNA. In some
embodiments, where the active agent is siRNA, the nanocarrier
further comprises a polycationic lipid. In some embodiments, the
polycationic lipid is 1,2-dioleoyl-3-trimethylammonium-propane
(DOTAP).
[0078] The "nanoparticle" of the invention is a particulate
carrier, a nanocapsule (NC) or a nanosphere (NS), which is
biocompatible and sufficiently resistant to chemical and/or
physical destruction, such that a sufficient amount of the
nanoparticles remains substantially intact after administration
into the human or animal body and for a sufficient period of time
to reach the desired target organ (tissue). Generally, the
nanoparticles are spherical in shape, having an average diameter of
up 2 .mu.m (micron) with the majority of the nanoparticles having
averaged diameter of less than 1 .mu.m (micron).
[0079] As indicated hereinabove, in some aspects and embodiments of
the invention, the nanoparticles have averaged diameter of less
than 1 micron. In some aspects and embodiments, the nanoparticles
have averaged diameter of between about 400 and 950 nm. In other
embodiments, the averaged diameter of said nanoparticle is between
about 400 and 900 nm. In some other embodiments, the averaged
diameter is between about 400 and 800 nm. In further embodiments,
the averaged diameter is between about 400 and 700 nm. In
additional aspects and embodiments, the nanoparticles have averaged
diameter of between 400 and 600 nm.
[0080] It should be noted that the averaged diameter of
nanoparticles may be measured by any method known to a person
skilled in the art. The term "averaged diameter" refers to the
arithmetic mean of measured diameters, wherein the diameters range
.+-.25%, .+-.15%, .+-.10%, or .+-.5% of the mean. Where the
nanoparticles are not spherical, the term refers to the effective
average diameter being the largest dimension of the particle.
[0081] The nanoparticle of the invention should be large enough to
be able to hold a plurality of nanocarriers, yet at the same time
be of a smaller enough size to be able to undergo
internalization.
[0082] The plurality (one or more) of "nanocarriers" which are
contained within the nanoparticles of the invention, are themselves
particulate carriers, each having an average diameter of less than
300, less than 250, or less than 200 nm. The nanocarriers may be in
the form of nanocapsules (NC) or nanospheres (NS). Generally, the
nanocarriers are spherical in shape. Where the shape of the
nanocarriers is not spherical, the diameter refers to the longest
dimension of the particle.
[0083] The number of nanocarriers which are encapsulated within a
single nanoparticle according to the invention may vary depending
on, e.g., the size of the nanocarrier or the relative sizes of the
nanocarrier and the nanoparticle. Typically, each nanoparticle may
contain between 1 and a few (6-7) dozens of nanocarriers (being
said plurality of nanocarriers). In some embodiments, each
nanoparticle comprises between 2 and 50 nanocarriers. In some
embodiments, each nanoparticle comprises between 2 and 40
nanocarriers. In some embodiments, each nanoparticle comprises
between 2 and 30 nanocarriers. In some embodiments, each
nanoparticle comprises between 2 and 20 nanocarriers. In some
embodiments, each nanoparticle comprises between 2 and 10
nanocarriers.
[0084] In some embodiments, each nanoparticle comprises more than 2
nanocarriers.
[0085] The nanocarriers are said to "contain" said at least one
active agent. As exemplified hereinbelow, the at least one active
agent may be contained in a core of said nanocarrier, and/or may be
contained in the material matrix making up the nanocarrier, and/or
may be associated with a surface region (one or more, or whole
surface) of said nanocarriers.
[0086] In some embodiments, where the nanocarriers are metallic
particles, e.g., gold nanospheres, the at least one active agent is
associated with a surface region (one or more, or whole surface) of
said metallic particles.
[0087] In some embodiments, the nanocarriers themselves are
prepared by nanospraying, as detailed herein.
[0088] In some embodiments, the averaged diameter of a nanocarrier
is at least about 50 nm.
[0089] In some embodiments, the averaged diameter of a nanocarrier
is between about 100 and 300 nm. In other embodiments, the averaged
diameter is between about 200 and 300 nm. In other embodiments, the
averaged diameter is between about 50 and 300 nm. In other
embodiments, the averaged diameter is between about 50 and 250 nm.
In further embodiments, the averaged diameter is between about 50
and 200 nm. In further embodiments, the averaged diameter is
between about 50 and 150 nm. In further embodiments, the averaged
diameter is between about 50 and 100 nm
[0090] As a person versed in the art would understand, the
"hydrophilicity" of the materials is a characteristic of materials
exhibiting affinity for water, while the "hydrophobic" materials
possess the opposite response to water.
[0091] The solubility or insolubility of the nanoparticles in
aqueous media may be altered by cross-linking the nanocarrier or
final nanoparticle material to increase or decrease the final
nanoparticle solubility in such media.
[0092] For the chosen application, the nanoparticle and/or the
nanocarrier may be in the form of a "nanocapsules", namely having a
core/shell structure, with a polymeric shell and a core which may
be empty or which may contain at least one oily or aqueous phase.
Alternatively the nanoparticles and/or nanocarriers may be of a
substantially uniform composition, namely as "nanospheres" (NSs) of
a continuous material, not featuring a distinct core/shell
structure.
[0093] In some embodiments, the nanoparticles of the invention and
the plurality of nanocarriers contained therein are in the form of
nanocapsules. In other embodiments, the nanoparticles and the
nanocarriers are both nanospheres. In some other embodiments, the
nanoparticles may be in the form of nanocapsules and the
nanocarriers may in the form of nanospheres. In further
embodiments, the nanoparticles may be in the form of nanospheres
and the nanocarriers may in the form of nanocapsules.
[0094] The term "encapsulation" (or any lingual variation thereof)
refers to, e.g., the containment of at least one nanocarrier within
a nanoparticle, or to the containment of an active material in a
nanocarrier (as defined hereinabove). Therefore, according to some
embodiments, the nanoparticles are said to encapsulate one or more
nanocarriers.
[0095] In some other embodiments, the nanoparticles encapsulate a
plurality of nanocarriers being selected from nanocapsules,
nanospheres and mixtures thereof. Notwithstanding the form of the
polarity of nanocarriers (NS and/or NC), the nanoparticles of the
invention may encapsulate a plurality of nanocarriers of different
materials and/or different active agents. The nanoparticles of the
invention, for example, may contain a plurality of nanocarriers of
the same polymeric material (thus having the same
hydrophilic/hydrophobic properties), with one or more different
active agents. Similarly, the nanoparticles of the invention may
contain a plurality of nanocarriers of different polymeric
materials, however containing each the same active agent.
[0096] In some aspects of the invention, there are provided
mixtures of nanoparticles of the invention, said mixtures
comprising one or more types of nanoparticles, said one or more
nanoparticles types differing from each other by at least any one
of:
[0097] 1. nanocarrier material,
[0098] 2. nanoparticle material,
[0099] 3. active agent,
[0100] 4. nanoparticle/nanocarrier form (NS or NC),
[0101] 5. nanoparticle/nanocarrier size.
[0102] As stated above, in some aspects and embodiments of the
invention, the nanoparticles are obtainable by nanosparying. This
process comprises transporting (e.g., delivering, spraying) through
a screen (e.g., mesh) having one or more orifices (holes, openings,
punctures, pinholes) of a predetermined size (diameter), a
colloidal composition comprising a plurality of nanocarriers and a
nanoparticle material e.g., a polymeric material, in a liquid
medium, said plurality of nanocarriers comprising at least one
active agent and said nanoparticle material is at least partially
soluble in said liquid medium, the size of said orifices
determining the (maximal) size (diameter) of the nanoparticles.
[0103] In some embodiments, the orifices allow production of
nanoparticles having averaged diameter of less than 4 micron, or
less than 2 micron, or less than 1 micron, or less than 950 nm, or
between 400 and 950 nm, or between about 400 and 900 nm, or between
about 400 and 800 nm, or between about 400 and 700 nm, or between
400 and 600 nm.
[0104] To permit such nanoparticle sizes, the orifices size (hole
diameter) is selected to range from 7 micron to 1 micron. In some
embodiments, the screen is between 4 and 6 micron mesh size. In
some embodiments, the screen is 4 micron mesh size.
[0105] In some embodiments, the colloidal composition is prepared
by mixing a plurality of nanocarriers with a nanoparticle material
e.g., a polymeric material, in a liquid medium.
[0106] In some embodiments, the nanospraying method further
comprises the step of drying the nanoparticles.
[0107] In some embodiments, the nanocarriers are obtained by
nanospraying.
[0108] The nanoparticles of the invention are mainly comprised of
polymers. The term "polymer" includes homopolymers, copolymers,
such as for example, block, graft, random and alternating
copolymers as well as terpolymers, further including their
derivatives, combinations and blends thereof. In addition to the
above, the term includes all geometrical configurations of such
structures including linear, block, graft, random, alternating,
branched structures, and combination thereof.
[0109] The polymers utilized in the construction of the
nanoparticles are biodegradable, namely, they degrade during in
vivo use. In general, degradation attributable to biodegradability
involves the degradation of a biodegradable polymer into its
component subunits, or digestion, e.g., by a biochemical process
carried out for example by enzymes, of the polymer into smaller,
non-polymeric subunits. The degradation may proceed in one or both
of the following: biodegradation involving cleavage of bonds in the
polymer matrix, in which case, monomers and oligomers typically
result, or the biodegradation involving cleavage of a bond internal
to side chain or that connects a side chain to the polymer
backbone. In some embodiments, biodegradation encompasses both
general types of biodegradation. The polymers are additionally
biocompatible, namely, they are substantially non-toxic or lacking
injurious impact on the living tissues or living systems to which
they come in contact with.
[0110] The nanoparticles of the invention may be used as a drug
delivery platform, enabling penetration and release of active
agents into targeted cells or organelles within the cell. To
facilitate cell membrane penetration, the nanoparticles of the
invention may be associated with different targeting agents.
[0111] In some embodiments, the outer surfaces of the nanoparticles
are associated with at least one targeting agent. In such
embodiments, the at least one targeting agent is selected from
monoclonal antibodies, such as trastuzumab (Herceptin.RTM.)
recognizing HER-2 receptor overexpressed in solid tumors; AMBLK8
recognizing H ferritin, cetuximab (Erbitux.RTM.) recognizing EGFR
receptors; Rituximab (MabThera.RTM.) recognizing CD20; bevacizumab
(Avastin.RTM.) inhibiting the function of a natural protein called
"vascular endothelial growth factor"
[0112] (VEGF) that stimulates new blood vessel formation;
ranibizumab (Lucentis.RTM.), providing stronger binding to VEGF-A;
small molecules such as folic acid or folate; hyaluronic acid or
hyaluronan; tumor penetrating peptides, typically about 6-15 kDa;
epidermal growth factor (EGF); transferrin; ferritin;
Arginine-Glycine-Aspartic acid (RGD) peptide; epithelial cell
adhesion molecule (EpCAM); intercellular adhesion molecule 1
(ICAM-1); carcinoembrionic antigen (CEA); vasoactive intestinal
peptide; CA 15-3 antigen; MUC1 protein; CD20; CD33; integrins;
lymphatic targeting moieties (such as LyP-1); aptamers, such as
PSMA aptamer or VEGF aptamer; oligosaccharides and others.
[0113] In some embodiments, the targeting agent is bevacizumab
(Avastin.RTM.) or Ranibizumab (Lucentis.RTM.).
[0114] As used herein, the term "association" or any lingual
variation thereof refers to the chemical or physical interaction
which holds two entities together (e.g., the nanoparticle and the
targeting agent, the nanocarrier surface with a linker moiety or
with an active agent, or any interaction referred to as such). The
interaction may be any type of chemical or physical bonding
interaction known to a person skilled in the art. Non-limiting
examples of such interactions (associations) include ionic bonding,
covalent bonding, coordination bonding, complexation, hydrogen
bonding, van der Waals bonding, hydrophobicity-hydrophilicity
interactions, etc. In some embodiments, the association is via
covalent bonding. In other embodiments, the association is via
coordinative bonding. It should be understood to a person skilled
in the art that in some cases the associative interactions between
two atoms or two chemical entities may involve more than one type
of chemical and/or physical interactions.
[0115] Once inside a living cell in vivo or in vitro, the active
agent has to be delivered to the proper organelle, or alternatively
escape compartmentalization into cell organelles, such as endosomes
and lysosomes, and be intracellularly bioavailable. Therefore, in
some embodiments, said at least one nanocarrier has a cationic
lipid (such as DOTAP) or cell penetrating peptides which contain
various amino acids, such as arginine or lysine residues,
conferring positive charges to the peptide. These peptides can
penetrate the cell and release the cargo of the nanocarrier in the
cytoplasm. Such peptides may be selected from HIV-TAT, penetratin,
Gramicidin S, MSI-103, MSI-103-Arg, PGLa, PGLa-Arg, Magainin 2,
Magainin-2-Arg, KIGAKI, BP100, MAP, MAP-Arg, SAP, PEP-1,
transportan, FP23 and others.
[0116] In some embodiments, the cationic lipid is DOTAP.
[0117] As noted above, the nanoparticles may comprise at least one
nanocarrier, the nanocarrier being a nanosphere comprising a
hydrophilic matrix (material), wherein the at least one active
agent is hydrophilic and distributed within the hydrophilic
matrix.
[0118] In some embodiments, the nanosphere hydrophilic material is
selected from dextran, hylauronate, human serum albumin (HSA) being
normal or cross-linked, bovine serum albumin (BSA) being normal or
cross-linked, chitosan, shellac, collagen, gelatin, gum arabic,
polyvinyl alcohol, cyclodextrin, each being alone or in combination
with one or more of the aforementioned. In other embodiments, the
hydrophilic material is human serum albumin (HSA), bovine serum
albumin (BSA) or hyaluronic acid.
[0119] In some embodiments, the hydrophilic material is human serum
albumin (HSA) having an average molecular weight of about 66,500
Da, or hyaluronic acid having an average molecular weight ranging
in size from 20,000 up to 1,000,000 Da.
[0120] According to such embodiments, the hydrophilic active agent
may be selected from therapeutic peptides or proteins, such as
exenatide, insulin, growth hormone, triptorelin acetate, buserelin,
nafarelin, and others; DNA, RNA, siRNA, tRNA or derivatives or
fragments thereof.
[0121] In some embodiments, the hydrophilic agent is siRNA.
[0122] In other embodiments, the siRNA has the nucleotide sequence
of SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12.
[0123] In some embodiments, the hydrophilic material is
cross-linked to lower the nanocarrier's solubility in aqueous
media.
[0124] In some embodiments, where the nanocarrier is hydrophilic in
nature, the nanoparticle may be in the form of a nanocapsule which
comprises a hydrophobic shell (i.e. a hydrophobic nanoparticle
encapsulating the hydrophilic nanocarriers). This arrangement
enables the encapsulation and stabilization of hydrophilic agents,
normally problematic to stabilize and deliver.
[0125] In such embodiments, the hydrophobic shell is of a polymer
selected from lactic acid, poly(D,L-lactic-co-glycolic acid)
(PLGA), poly(D,L-lactic acid) (PLA), poly(.epsilon.-caprolactone),
poly(2-dimethylamino-ethylmethacrylate) homopolymer,
poly(2-dimethylamino-ethylmethacrylate)-b-poly(ethyleneglycol)-.alpha.-me-
thoxy-w-metacrylate copolymers, polycyanoacrylates, polyanhydride
polymers and combinations thereof.
[0126] In some embodiments, the hydrophobic shell is a PEGylated
derivative of a polymer selected from lactic acid,
poly(D,L-lactic-co-glycolic acid) (PLGA), poly(D,L-lactic acid)
(PLA), poly(.epsilon.-caprolactone),
poly(2-dimethylamino-ethylmethacrylate) homopolymer,
poly(2-dimethylamino-ethylmethacrylate)-b-poly(ethyleneglycol)-.alpha.-me-
thoxy-w-metacrylate copolymers, polycyanoacrylates, polyanhydride
polymers and combinations thereof.
[0127] In other embodiments, the hydrophobic shell is selected from
lactic acid, poly(D,L-lactic-co-glycolic acid) (PLGA) and
combinations thereof, including PEGylated derivatives thereof
(alone or in combination). In such embodiments, the PLGA has a
molecular weight of between about 4,000 and 100,000 Da.
[0128] In order to decrease cell membrane deterrence, the
hydrophobic shell may have an outer surface associated with at
least one polyethylene glycol (PEG) moiety.
[0129] In other embodiments of the invention, the nanoparticles may
comprise at least one nanocarrier, the nanocarrier being a
nanosphere comprising a hydrophobic polymer matrix; the at least
one active agent contained within said nanosphere is also
hydrophobic.
[0130] In such embodiments, the hydrophobic polymer matrix may be
selected from lactic acid, poly(D,L-lactic-co-glycolic acid)
(PLGA), poly(D,L-lactic acid) (PLA), poly(.epsilon.-caprolactone),
poly(2-dimethylamino-ethylmethacrylate) homopolymer,
poly(2-dimethylamino-ethylmethacrylate)-b-poly(ethyleneglycol)-.alpha.-me-
thoxy-w-metahcrylate copolymers, polycyanoacrylates and
combinations thereof and their PEGylated derivatives.
[0131] In other embodiments, the hydrophobic polymer matrix is
selected from lactic acid, poly(D,L-lactic-co-glycolic acid) (PLGA)
and combinations thereof, including mixtures with PEGylated
derivatives thereof.
[0132] In some other embodiments, said PLGA has a molecular weight
of between about 4,000 and 100,000 Da.
[0133] In further embodiments, the hydrophobic active agent is
selected from an analgesic or anti-inflammatory agent (such as
aloxiprin, auranofin, azapropazone, benorylate, diflunisal,
etodolac, fenbufen, fenoprofen calcim, flubiprofen, ibuprofen,
indomethacin, ketoprofen, meclofenamic acid, mefenamic acid,
nabumetone, naproxen, oxyphenbutazone, phenylbutazone, piroxican,
or sulindac); an enthelmintic agent (such as albendazole, bephenium
hydroxynaphthoate, cambensazole, dichlorophen, ivermectin,
mebendazole, oxamniquine, oxefendazole, oxantel embonate,
praziquantel, pyrantel embonate, or thiabendazole); an
anti-arrhythmic agent (such as amiodarone, disopyramide, flecainide
acetate, or quinidine sulphate); an anti-bacterial agent (such as
benethamine penicillin, cinoxacin, ciprofloxacin, clarithromycin,
clofazimine, cloxacillin, demeclocycline, doxycycline,
erythromycin, ethionamide, imipenem, nalidixic acid,
nitrofurantoin, rifampicin, spiramycin, sulphabenzamide,
sulphadoxine, sulphacetamide, sulphamerazine, sulphadiazine,
sulphafurazole, sulphamethoxazole, sulphapyridine, tetracycline, or
trimethoprim); an anti-coagulant (such as dicoumarol, dipyridamole,
nicoumalone or phenindione); an anti-depressant (such as amoxapine,
meprotiline, mianserin, nortriptyline, trazodone, or tirimipramine
maleate); an antidiabetic (such as acetothexamide, chlorpropamide,
glibenclamide, gliclazide, glipizide, tolazamide, or tolbutramide);
an anti-epileptic (such as beclamide, carbamazepine, clonazepine,
ethotoin, methoin, methsuximide, methylphenobarbitone,
oxcarbazepine, paramethadione, phenacemide, phenobarbitone,
phenyloin, phensuximide, primidone, sulthiame, or valproic acid);
an anti-fungal agent (such as amphotericin, butoconazole nitrate,
clotrimazole, econazole nitrate, fluconazole, flucytosine,
griseofulvin, itraconazole, ketoconazole, miconazole, natamycin,
nystatin, sulconazole nitrate, terbinafine, terconazole,
tioconazole or undecenoic acid); an anti-gout agent (such as
allopurinol, probenecid or sulphin-pyrazone); an anti-hypertensive
agent (such as amlodipine, benidipine, darodipine, dilitazem,
diazoxide, felodipine, guanabenz acetate, isradipine, minoxidil,
nicardipine, nifedipine, nimodipine, phenoxybenzamine, prazosin,
reseprine or terazosin); an anti-malarial agent (such as
amodiaquine, chloroquine, chlorproguanil, halofantrine, mefloquine,
proganil, pyrimethamine, or quinine sulphate); an anti-migraine
agent (such as dihydroergotamine mesylate, ergotamine tartarate,
methysergide maleate, pizotifen maleate or sumatriptan succinate);
an anti-muscarinic agent (such as atropine, benzhexol, biperiden,
ethopropazine, hyoscyamine, mepenzolate bromide, oxyphencylcimine,
or tropicamide); an anti-neuroplastic agent or immunosuppressant
(such as aminoglutethimide, amsacrine, azathioprine, busulphan,
chlorambucil, cyclosporin, dacarbazine, estramustine, etoposide,
lomustine, melphalan, mercaptopurine, methotrexate, mitomycin,
mitotane, mitozantrone, procarbazine, tamoxifen citrate,
testolactone, tacrolimus, or sirolimus); an anti-protazoal agent
(such as benznidazole, clioquinol, decoquinate,
diiodohydroxyquinoline, diloxanide furoate, dinitolmide,
furzolidone, metronidazole, nimorazole, nitrofurazone, ornidazole,
or tinidazole); an anti-thyroid agent (such as carbimazole or
propylthiouracil); an alixiolytic, sedative, hypnotic or
neuroleptic agent (such as alparzolam, amylobarbitone, barbitone,
bentazepam, bromazepam, bromperidol, brotizolam, butobarbitone,
carbromal, chlordiazepoxide, chlormethiazole, chlorpromazine,
clobazam, clotiazepam, clozapine, diazepam, droperidol, ethinamate,
flunanisone, flunitrazepam, fluopromazine, flupenthixol decanoate,
fluphenazine decanoate, flurazepam, baloperidol, lorazepam,
lormetazepam, medazepam, meprobamate, methaqualone, midazolam,
nitrazepam, oxazepam, pentobarbitone, perphenazine primozide,
prochlorperazine, sulpiride, temazepam, thioridazine, triazolam, or
zopiclone); a beta-blocker (such as acebutolol, alprenolol,
atenolol, labetalol, metoprolol, nadolol, oxprenolol, pindolol, or
propranolol); a cardiac inotropic agent (such as amrinone,
digitoxin, digoxin, enoximone, lanatoside C, or medigoxin); a
corticosteroid (such as beclomethasone, betamethasone, budesonide,
cortisone acetate, desoxymethasone, dexamethasone, fludrocortisones
acetate, flunisolide, flucortolone, fluticasone propionate,
hydrocortisone, methylprednisolone, prednisolone, prednisone, or
triamcinolone); a diuretic agent (such as acetazolamide, amiloride,
bendofluazide, bumetanide, chlorothiazide, chlorthalidone,
ethacrynic acid, frusemide, metolazone, spironolactone, or
triamterene); an anti-Parkinsonian agent (such as bromocriptine
mesylate, or lysuride maleate); a gastro-intestinal agent (such as
bisacodyl, cimetidine, cisapride, diphenoxylate, domperidone,
famotidine, loperamide, mesalazine, nizatidine, omeprazole,
ondansetron, ranitidine, or sulphasalazine); an histamine
H1-receptor antagonist (such as acrivastine, astemizole,
cinnarizine, cyclizine, cyproheptadine, dimenhydrinate,
flunarizine, loratadine, meclozine, oxatomide or terfenadine); a
lipid regulating agent (such as bezafibrate, clofibrate,
fenofibrate, gemfibrozil, or probucol); a nitrate or anti-anginal
agent (such as amyl nitrate, glyceryl trinitrate, isosorbide
dinitrate, isosorbide mononitrate, or pentaerythritol
tetranitrate); a nutritional agent (such as betacarotene, vitamin
A, vitamin B2, vitamin D, vitamin E or vitamin K); an HIV protease
inhibitor (such as nelfinavir); an opioid analgesic (such as
codeine, dextropropyoxyphene, diamorphine, dihydrocodeine,
meptazinol, methadone, morphine, nalbuphine, or pentazocine); a sex
hormone (such as clomiphene citrate, danazol, ethinyl estradiol,
medroxyprogesterone acetate, mestranol, methyltestosterone,
morethisterone, norgestrel, estradiol, conjugated oestrogens,
progesterone, stanozolol, stibestrol, testosterone, or tibolone);
or a stimulant agent (such as amephetamine, dexamphetamine,
dexfenfluramine, fenfluramine or mazindol).
[0134] In some embodiments where the nanocarrier is of a
hydrophobic nature, the nanoparticle may be a nanocapsule
comprising a hydrophilic shell. In such embodiments, the
hydrophilic shell may be selected from dextran, hylauronate, human
serum albumin (HSA), bovine serum albumin (BSA), chitosan, shellac,
collagen, gelatin, gum arabic, polyvinyl alcohol, cyclodextrin,
alone or in combination. If needed, each of said polymers may be
cross-linked.
[0135] According to some embodiments, the hydrophilic material is
human serum albumin (HSA), bovine serum albumin (BSA) or hyaluronic
acid.
[0136] According to other embodiments, the hydrophilic matrix is
human serum albumin (HSA) having an average molecular weight of
about 66,500 Da.
[0137] The drug delivery systems of the invention may be tailored
and modified based on the active agent (material) to be carried
therein. The nanoparticles carrying a plurality of nanocarriers may
be used to carry and deliver one or more active materials. For
example, a hydrophobic active material may be encapsulated in a
hydrophobic nanocarrier (nanosphere or nanocapsule) and a further
active material which is hydrophilic in nature may be entrapped
within the hydrophilic material matrix of the nanoparticle
material. Similarly, hydrophilic nanocarriers may encapsulate more
than one hydrophilic active materials and the hydrophobic
nanoparticle material may hold one or more hydrophobic active
materials.
[0138] Depending on the final application and/or the nature of the
delivery system, whether the active agent is hydrophobic or
hydrophilic, it can be associated to the primary nanocarrier (being
hydrophobic or hydrophilic), via chemical bonds (as defined
hereinabove, e.g., polar, ionic, van der Waals, etc) and for
polyanionic macromolecules, via the addition of a `helper lipid`
(such as DOTAP). This chemical association of the active agent to
the nanocarriers or nanoparticles prevents leaking of the active
agent and the efficacy of encapsulation process is maintained or
secured (this approach may be useful depending on the final
application and/or particular delivery system).
[0139] The "active agent" to be encapsulated within the plurality
of nanocarriers or in the polymeric matrix of the nanocarriers
and/or nanoparticles may be selected amongst vitamins, proteins,
anti-oxidants, nucleic acids, short or long oligonucleotides (in
different conformations), siRNA and its chemical derivatives,
peptides, polypeptides, lipids, carbohydrates, hormones,
antibodies, monoclonal antibodies, vaccines and other prophylactic
agents, drugs, diagnostic agents, contrasting agents, nutraceutical
agents, small molecules (of a molecular weight of less than about
1,000 Da or less than about 500 Da), electrolytes, immunological
agents and any combination of any of the aforementioned.
[0140] In some additional embodiments, particular agents to be
encapsulated in systems according to the invention include
exenatide, insulin and others.
[0141] In other embodiments, the active agent is siRNA.
[0142] In further embodiments, the siRNA to be encapsulated is
selected from siRNA having any one of the nucleotide sequences of
SEQ ID NOS: 1 through 12.
[0143] In order to modify the hydrophibicity/hydrophilicity of a
certain active material, the material may be appended with a
negative or positive charge or with a lipophilic (hydrophobic)
moiety.
[0144] In some embodiments, said active agent is negatively charged
and the nanocarrier is also negatively charged. In such
embodiments, the cationic lipid is selected from
1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), stearylamine, and
oleylamine.
[0145] According to some embodiments, the cationic lipid is
DOTAP.
[0146] In some embodiments, where the active agent is associated
with at least a region of the nanocarrier surface, the association
(as generally defined above) may be directly to chemical groups
present on the surface of the nanocarrier particle, or may be via
one or more linking groups which chemically or physically associate
the surface region with the active material. The linking group may
be a single atom or a group of atoms and may be selected in a
non-limiting fashion from thiols, hydroxides, amines, alkyl groups,
phosphates, carboxylates, PEGs, and other known in the art.
[0147] The invention provides a nanoparticle encapsulating a
plurality of nanocarriers (one or more), at least one of said
plurality of nanocarriers containing at least one active agent, the
active agene being siRNA.
[0148] In some embodiments, the nanocarrier further comprises a
polycationic lipid, the polycationic lipid being
1,2-dioleoyl-3-trimethylammonium-propane (DOTAP).
[0149] In other embodiments, said nanoparticle has an averaged
diameter of between 400 and 950 nm.
[0150] In some other embodiments, the nanoparticle material is
hydrophobic. In such embodiments, the nanoparticle material is
selected from PLA, PLGA and mixtures thereof.
[0151] In another one of its aspects, the invention provides a
composition comprising a plurality of nanoparticles, as disclosed
herein. In some embodiments, the composition is a pharmaceutical
composition, and further comprising a pharmaceutically acceptable
carrier.
[0152] The "pharmaceutically acceptable carriers" described herein,
for example, vehicles, adjuvants, excipients, or diluents, are well
known to those who are skilled in the art and are readily available
to the public. It is preferred that the pharmaceutically acceptable
carrier be one which is chemically inert to the active compounds
and one which has no detrimental side effects or toxicity under the
conditions of use.
[0153] The choice of carrier will be determined in part by the
particular active agent, as well as by the particular method used
to administer the composition. Accordingly, there is a wide variety
of suitable formulations of the pharmaceutical composition of the
present invention. The following formulations for oral, parenteral,
intravenous, intramuscular, or intraperitoneal administration are
merely exemplary and are in no way limiting.
[0154] The pharmaceutical composition may be adapted for
administration by a variety of routes including oral, rectal,
vaginal, subcutaneous, intravenous, intramuscular, pulmonary,
topical or dermal, eye drops and intranasal. Such pharmaceutical
composition is prepared in a manner well known in the
pharmaceutical art. In making the pharmaceutical composition of the
invention, the aforementioned components are usually mixed with an
excipient, diluted by an excipient or enclosed within such a
carrier which can be manipulated to the desired form. Based on the
particular mode of administration, the pharmaceutical composition
may be formulated into tablets, pills, capsules, sachets, granules,
powders, chewing gum, suspensions, emulsions, creams, ointments,
anhydrous or hydrous topical formulations and solutions.
[0155] The pharmaceutically acceptable carriers, for example,
vehicles, adjuvants, excipients, or diluents, are well-known to
those who are skilled in the art and are readily available to the
public. It is preferred that the pharmaceutically acceptable
carrier be one which is chemically inert to the active formulation
and each of its components and one which has no detrimental side
effects or toxicity under the conditions of use.
[0156] The choice of carrier will be determined in part by the
particular formulation of the invention, as well as by the
particular method used to administer the composition. Accordingly,
there is a wide variety of suitable formulations of the
pharmaceutical composition of the present invention.
[0157] In some embodiments, the pharmaceutical composition is
adapted as a delivery system for transporting a therapeutic agent,
orally, parenterally or intravenous into the circulatory system
(cardiovascular system) of a subject.
[0158] Formulations suitable for oral administration can consist of
(a) liquid solutions, such as an effective amount of the
nanoparticles, or composition comprising same, dissolved in
diluents, such as water, saline, or juice (e.g. orange juice); (b)
capsules, sachets, tablets, lozenges, and troches, each containing
a predetermined amount of the active ingredient, as solids or
granules;
[0159] (c) powders; (d) suspensions in an appropriate liquid; and
(e) suitable emulsions. Liquid formulations may include diluents,
such as water and alcohols, for example, ethanol, benzyl alcohol,
and the polyethylene alcohols, either with or without the addition
of a pharmaceutically acceptable surfactant, suspending agent, or
emulsifying agent. Capsule forms can be of the ordinary hard- or
soft-shelled gelatin type containing, for example, surfactants,
lubricants, and inert fillers, such as lactose, sucrose, calcium
phosphate, and corn starch. Tablet forms can include one or more of
lactose, sucrose, mannitol, corn starch, potato starch, alginic
acid, microcrystalline cellulose, acacia, gelatin, guar gum,
colloidal silicon dioxide, talc, magnesium stearate, calcium
stearate, zinc stearate, stearic acid, and other excipients,
colorants, diluents, buffering agents, disintegrating agents,
moistening agents, preservatives, flavoring agents, and
pharmacologically compatible carriers. Lozenge forms can comprise
the active ingredient in a flavor, usually sucrose and acacia or
tragacanth, as well as pastilles comprising the active formulation
in an inert base, such as gelatin and glycerin, or sucrose and
acacia, emulsions, gels, and the like containing, in addition to
the active formulation, such carriers as are known in the alt
[0160] The parenteral formulations will typically contain from
about 0.5 to about 25% by weight of the active ingredient in
solution. Suitable preservatives and buffers can be used in such
formulations. In order to minimize or eliminate irritation at the
site of injection, such compositions may contain one or more
nonionic surfactants having a hydrophile-lipophile balance (HLB) of
from about 12 to about 17. The quantity of surfactant in such
formulations ranges from about 5 to about 15% by weight. Suitable
surfactants include polyethylene sorbitan fatty acid esters, such
as sorbitan monooleate and the high molecular weight adducts of
ethylene oxide with a hydrophobic base, formed by the condensation
of propylene oxide with propylene glycol. The parenteral
formulations can be presented in unit-dose or multi-dose sealed
containers, such as ampules and vials, and can be stored in a
freeze-dried (lyophilized) condition requiring only the addition of
the sterile liquid carrier, for example, water, for injections,
immediately prior to use. Extemporaneous injection solutions and
suspensions can be prepared from sterile powders, granules, and
tablets of the kind previously described.
[0161] The compounds of the present invention may be made into
injectable formulations. The requirements for effective
pharmaceutical carriers for injectable compositions are well known
to those of ordinary skill in the art. See Pharmaceutics and
Pharmacy Practice, J. B. Lippincott Co., Philadelphia, Pa., Banker
and Chalmers, eds., pages 238-250 (1982), and ASHP Handbook on
Injectable Drugs, Toissel, 4.sup.th ed., pages 622-630 (1986).
[0162] In some embodiments, the delivery system is adaptable for a
facilitated targeted therapeutic delivery and controlled release
administration of a therapeutically effective amount of the active
agent.
[0163] As known, the "effective amount" for purposes herein may be
determined by such considerations as known in the art. The amount
must be effective to achieve the desired therapeutic effect,
depending, inter alia, on the type and severity of the disease to
be treated and the treatment regime. The effective amount is
typically determined in appropriately designed clinical trials
(dose range studies) and the person versed in the art will know how
to properly conduct such trials in order to determine the effective
amount. As generally known, the effective amount depends on a
variety of factors including the affinity of the ligand to the
receptor, its distribution profile within the body, a variety of
pharmacological parameters such as half life in the body, on
undesired side effects, if any, on factors such as age and gender,
and others. The nanoparticle containing active material(s)
according to the invention may be used as such to induce at least
one effect, e.g., "therapeutic effect", or may be associated in
conjugation with at least one other agent to induce, enhance,
arrest or diminish at least one effect or side effect, by way of
treatment or prevention of unwanted conditions or diseases in a
subject. The at least one other agent (substance, molecule,
element, compound, entity, or a combination thereof) may be
selected amongst therapeutic agents, i.e., agents capable of
inducing or modulating a therapeutic effect when administered in a
therapeutically effective amount, and non-therapeutic agents, i.e.,
which by themselves do not induce or modulate a therapeutic effect
but which may endow the nanoparticles with a selected
characteristic, as will be further disclosed hereinbelow.
[0164] The pharmaceutical composition of the present invention may
be selected to treat, prevent or diagnose any pathology or
condition, depending on the active material contained within the
nanoparticles. The term "treatment" or any lingual variation
thereof, as used herein, refers to the administering of a
therapeutic amount of the composition or system of the present
invention which is effective to ameliorate undesired symptoms
associated with a disease, to prevent the manifestation of such
symptoms before they occur, to slow down the progression of the
disease, slow down the deterioration of symptoms, to enhance the
onset of remission period, slow down the irreversible damage caused
in the progressive chronic stage of the disease, to delay the onset
of said progressive stage, to lessen the severity or cure the
disease, to improve survival rate or more rapid recovery, or to
prevent the disease from occurring or a combination of two or more
of the above.
[0165] In another aspect, the invention also provides a kit or a
commercial package containing the composition of the invention as
herein described, and instructions for use. In some embodiments,
the composition of the invention or a fraction derived therefrom
may be present in the kit in separate compartments or vials.
[0166] The kit may further comprise at least one carrier, diluent
or solvent useful for the dissolution of the active components, the
dilution thereof or generally for the preparation of the
composition. The composition may be prepared by the end user (the
consumer or the medical practitioner) according to the instructions
provided or the experience and/or training of the end-user.
[0167] The kit may also comprise measuring tools for measuring the
weight, volume or concentration of each component (active
composition and/or carriers).
[0168] In another one of its aspects, the invention provides a
process for obtaining the nanoparticle of the invention, as herein
described, the process comprising: [0169] obtaining at least one
nanocarrier, said nanocarrier comprising at least one active agent;
and [0170] encapsulating said at least one nanocarrier into a
nanoparticle.
[0171] Depending on the nature of the active agent (material) or
plurality thereof to be contained in the system of the invention,
the process of the invention may be carried out in a variety of
equivalent forms. In general, the process comprises: [0172]
selecting a polymer having hydrophobic or hydrophilic properties on
the basis of whether the active agent is hydrophobic or
hydrophilic, respectively; [0173] dissolving the polymer and the
active agent in a liquid medium; [0174] treating said liquid medium
comprising the polymer and active agent with a further liquid
(where the liquid medium is organic, the further liquid is aqueous,
and vice versa); and [0175] isolating said nanocarriers.
[0176] In some embodiments, where the active agent is hydrophobic,
the nanocarrier material is hydrophobic and the nanoparticle
material is hydrophilic, the process for preparing the
nanoparticles of the invention comprises: [0177] dissolving a
hydrophobic polymer in an optionally water-miscible organic solvent
to form an organic phase; said organic solvent being selected, in
some embodiments from ethanol, methanol, chloroform dichloromethane
(DCM), diethyl ether, acetone and acetonitrile (ACN); [0178]
contacting said organic phase with an aqueous phase, the aqueous
phase optionally comprising a surfactant, to thereby obtain said
nanocarriers; and [0179] incubating said nanocarriers with a
solution of said active agent to allow association of said active
agent with at least a portion of the surface of said
nanocarriers.
[0180] In some embodiments, the organic miscible solvent is
selected from ethanol, methanol, chloroform dichloromethane (DCM),
diethyl ether, acetone and acetonitrile (ACN).
[0181] As used herein, the term "contacting", or any lingual
variation thereof, refers to the bringing together of the organic
phase and the aqueous phase in such a way to allow intimate contact
between them.
[0182] It should be noted, that the term "solution" should be given
its broadest definition to encompass a liquid state in which one
component is fully dissolved in another or in a liquid medium, a
liquid state of emulsion (nano- or microemulsion) of one or more
components of the precursor solution in another or in a medium, and
a state of dispersion (nano- or microdispersion) of one or more
components of the precursor solution in another or in a medium.
[0183] In some embodiments, where the active agent is hydrophilic,
the nanocarrier material is hydrophilic and the nanoparticle
material is hydrophobic, the process comprises: [0184] dissolving a
hydrophilic polymer in an aqueous solution of the active agent to
form an aqueous phase; and [0185] continuously adding an organic
phase comprising a desolvating agent to the aqueous phase under a
pH permitting the formation said nanocarriers, the active agent
being distributed within the nanocarrier.
[0186] In some embodiment, the process optionally comprises
cross-linking said hydrophilic polymer matrix.
[0187] In other embodiments, said pH is between 6 and 9. In some
embodiments, the pH is 7.
[0188] In further embodiments, the desolvating agent is selected
from acetone or ethanol, and acetonitrile.
[0189] The organic phase used in the process of the invention
further comprises a positively charged lipid (cationic lipid), as
define herein.
[0190] In all methods of preparing the nanoparticles of the
invention, the final step of forming the nanoparticle coating
around a plurality of nanocarriers may be achieved by nanospraying
the nanocarriers into a solution comprising the nanoparticle
polymer material. In accordance with other embodiments, said
nanoparticles are obtained by encapsulation conducted in a
nano-spray dryer.
[0191] In some embodiments, the process of the invention further
comprises functionalizing at least a portion of the outer surface
of said nanoparticle with at least one targeting moiety. In other
embodiments, said at least a portion is the entire surface of the
nanoparticle.
[0192] In some embodiments, said at least one targeting moiety is
selected from PEG, trastuzumab (Herceptin.RTM.) recognizing HER-2
receptor overexpressed in solid tumors; AMBLK8 recognizing H
ferritin; cetuximab (Erbitux.RTM.) recognizing EGFR receptors;
Rituximab (MabThera.RTM.) recognizing CD20; bevacizumab
(Avastin.RTM.) inhibiting the function of a natural protein called
"vascular endothelial growth factor" (VEGF) that stimulates new
blood vessel formation; ranibizumab (Lucentis.RTM.), providing
stronger binding to VEGF-A; small molecules such as folic acid or
folate; hyaluronic acid or hyaluronan; tumor penetrating peptides,
typically about 6-15 kDa; epidermal growth factor (EGF);
transferrin; ferritin; Arginine-Glycine-Aspartic acid (RGD)
peptide; epithelial cell adhesion molecule (EpCAM); intercellular
adhesion molecule 1 (ICAM-1); carcinoembrionic antigen (CEA);
vasoactive intestinal peptide; CA 15-3 antigen; MUC1 protein; CD20;
CD33; integrins; lymphatic targeting moieties (such as LyP-1);
aptamers, such as PSMA aptamer or VEGF aptamer; oligosaccharides
and others.
[0193] According to some embodiments, the targeting agent is
bevacizumab (Avastin.RTM.) or ranibizumab (Lucentis.RTM.).
BRIEF DESCRIPTION OF THE DRAWINGS
[0194] In order to understand the disclosure and to see how it may
be carried out in practice, embodiments will now be described, by
way of non-limiting example only, with reference to the
accompanying drawings, in which:
[0195] FIGS. 1A-1B are schematic diagrams of the nanospray dryer
principle of action (FIG. 1B) and a nanoparticle obtained therefrom
(FIG. 1A).
[0196] FIGS. 2A-2B are integrity evaluations by gel retardation
assay (PAGE 8%) for GFP-siRNA from ultrafiltrate of washed PLGA NS
s, after 1 hr of incubation. FIG. 2A: ultrafiltrate from
formulations A and B--Lane No. 1--ladder, Lane No. 2 with 100 ng
GFP-siRNA as control. Lanes 3, 4 are with 10 .mu.l ultrafiltrate of
A1 and A2 respectively. Lanes 5, 6 are with 4 .mu.l ultrafiltrate
of A3 and A4 respectively. Lanes 7, 8 are with 10 .mu.l
ultrafiltrate of B1 and B2 respectively. Lanes 9, 10 are with 4
.mu.l ultrafiltrate of B3 and B4 respectively. FIG. 2B:
ultrafiltrate from formulations D and C: Lane No. 1--ladder, Lane
No. 2 with 100 ng GFP-siRNA as control. Lanes 3, 4 are with 16
.mu.l ultrafiltrate of C1 and C2 respectively. Lanes 5, 6 are with
16 .mu.l ultrafiltrate of C3 and C4 respectively. Lanes 7, 8 are
with 16 .mu.l ultrafiltrate of D1 and D2 respectively. Lanes 9, 10
are with 16 .mu.l ultrafiltrate of D3 and D4 respectively. In all
samples numbers 1,2 stands for duplicates from the formulation
incubated with 50 .mu.g GFP-siRNA and numbers 3,4 stands for
duplicates from the formulation incubated with 100 .mu.g
GFP-siRNA.
[0197] FIGS. 3A-3B are SEM characterizations of highly diluted
crosslinked HSA NSs (mean size 58.+-.30 nm, ZP -38.+-.9).
[0198] FIGS. 4A-4B are SEM characterizationzs of highly diluted
crosslinked HSA NSs with 0.03 mg DOTAP, encapsulating siRNA (FIG.
4A: NSs with Chol-GFP-siRNA, FIG. 4B: NPs with GFP-siRNA).
[0199] FIG. 5 is an HPLC chromatogram for GFP-siRNA extracted from
crosslinked HSA NSs made with 0.03 mg DOTAP and 200 .mu.g siRNA at
pH 8 and untreated GFP-siRNA control, injected at the same
gradient. The thick line is for absorbance at 260 nm and the thin
line for absorbance at 280 nm. For siRNA (unlike proteins or
peptides) the ratio A.sub.2601280 is in the range of 1.8 to 2. The
peak at app. 7.5 is attributed to the presence of GFP-siRNA
extracted from crosslinked HSA NSs.
[0200] FIG. 6 is an HPLC chromatogram for Chol-GFP-siRNA extracted
from crosslinked HSA NSs made with 0.03 mg DOTAP and 200 .mu.g
siRNA at pH 8 and untreated Chol-GFP-siRNA control, injected at the
same gradient. The thick line is for absorbance at 260 nm and the
thin line for absorbance at 280 nm. For siRNA (unlike proteins or
peptides) the ratio A.sub.2601280 is in the range of 1.8 to 2. The
peak at app. 25.0 is attributed to the presence of Chol-GFP-siRNA
extracted from crosslinked HSA NSs.
[0201] FIGS. 7A-7B are SEM micrographs of Dextran NCs encapsulating
PLGA NSs. The NCs produced by the spray drying process of 0.4%
(w/v) Dextran 40 in DDW (40 mg) consists of 30 mg of PLGA NSs
(.about.100 nm, ZP of -33 mV).
[0202] FIGS. 8A-8H are SEM micrographs of HSA NCs encapsulating
primary PLGA NSs. The NCs produced by the spray drying process of
HSA in different % (w/v) in DDW consist of PLGA NSs (.about.100
nm). FIGS. 8A-80B: 1.6% HSA (200 mg) encapsulating 30 mg of
negatively charged NSs of PLGA (ZP-33 mV). FIGS. 8C-8D: 0.75% HSA
(56 mg) encapsulating 15 mg of positively charged NSs of PLGA
(ZP+66 mV). FIGS. 8E-8F: 0.5% HSA (56 mg) encapsulating 14 mg of
negatively charged NSs of PLGA (ZP-33 mV). FIGS. 8G-8H: 0.25% (28
mg) HSA encapsulating 7 mg of negatively charged NSs of PLGA (ZP-33
mV).
[0203] FIGS. 9A-9B are SEM micrographs of PEG-PLGA NCs
encapsulating primary crosslinked HSA NSs. The NCs prepared by the
spray drying process of PEG-PLGA (10.5 mg) in acetone consist of
1.6 mg HSA NSs (.about.100 nm, ZP-51 mV). During the spraying
process appearance of crust on the spraying head was observed,
leading to fusion of the NCs.
[0204] FIGS. 10A-10B are SEM micrographs of PLGA NCs encapsulating
primary crosslinked HSA NSs. The NCs prepared by the spray drying
process of PLGA (9.8 mg) in acetone consist of 1.6 mg HSA NSs
(.about.100 nm, ZP-51 mV).
[0205] FIGS. 11A-11B are SEM micrographs of (A) PLGA NCs
encapsulating 1.6 mg of primary crosslinked HSA NPs and of (B)
PEG-PLGA NCs encapsulating 1.6 mg of primary
[0206] HSA NSs and their element analysis using EDS (Energy
Dispersive X-ray Spectroscopy). The nitrogen can originate only
from the HSA NSs.
[0207] FIGS. 12A and 12B are SEM micrographs of polymeric NCs
encapsulating primary crosslinked HSA NPs. FIG. 12A: PLGA NCs, FIG.
12B: PEG-PLGA NCs. The NCs made by spray drying process of polymer
(16 mg) in acetonitrile consist of 10 mg crosslinked HSA NSs
(.about.100 nm, ZP-43 mV).
[0208] FIGS. 13A-13B are size distribution and SEM micrographs of
sample AO-57: PLGA (50 kDa) NCs encapsulating of primary
crosslinked HSA NSs. FIG. 13C are NCs dispersed in water after 4
days FIGS. 14A-14B are size distribution and SEM micrographs of
sample AO-66: PLGA (50 kDa) NCs encapsulating of primary
crosslinked HSA NSs loaded with GFP-siRNA. The NCs made by spray
drying process of polymer (42 mg) in 40 ml acetonitrile consist of
12.6 mg crosslinked HSA NSs (.about.100 nm, ZP-43 mV).
[0209] FIGS. 15A-15B are size distribution and SEM micrographs of
sample AO-68: PLGA (50 kDa) NCs encapsulating of primary
crosslinked HSA NSs loaded with Chol-GFP-siRNA. The NCs made by
spray drying process of polymer (64 mg) in 60 ml acetonitrile
consist of 15 mg crosslinked HSA NSs (.about.100 nm, ZP-43 mV).
[0210] FIGS. 16A-16B are gel retardation assays (PAGE 8%) for
evaluation of free siRNA integrity after exposure to different
conditions. Integrity evaluation was made for GFP-siRNA (FIG. 16A)
and Chol-GFP-siRNA (FIG. 16B). Lane No. 1--ladder, Lane No. 2,
3--controls with 100 and 50 ng of untreated siRNA respectively.
Lanes 4, 6 and 8, with siRNA exposed to pH 7, 8 and 9,
respectively. Lanes 5, 7 and 9, with siRNA exposed to pH 7, 8 and 9
in the presence of glutaraldehyde (0.014% (v/v)), respectively.
[0211] FIGS. 17A-17B are gel retardation assays (PAGE 8%) for
evaluation of extracted siRNA. Integrity evaluation was made for
GFP-siRNA (FIG. 17A) and Chol-GFP-siRNA (FIG. 17B). Lane No.
1--ladder, Lane No. 2--control with 100 ng of untreated siRNA.
Lanes 3-8 with siRNA extracted from primal crosslinked HSA NSs
(produced in different pH conditions), Lanes 9 and 10, with siRNA
extracted from NCs loaded with the primal crosslinked HSA.
[0212] FIG. 18A is a SEM imaging of BSA NSs following preparation
and washings using the Vivaspin technique. The NSs were dried at
room temperature on a slide following spreading of one droplet of
the dispersion; FIG. 18B is a SEM imaging of BSA NSs following
preparation and washings using the Vivaspin technique. The NSs were
dried at room temperature on a slide following spreading of one
droplet of the dispersion.
[0213] FIGS. 19A-19B show uptake of 2% FITC-labeled HSA NPs in
A-431 cells using CLSM. Uptake after 4 h (FIG. 19A) and 22 h (FIG.
19B) of incubation at 37.degree. C. NPs concentration is 2 mg/ml
(1.5 ml per well).
DETAILED DESCRIPTION
[0214] In the present invention, double nanoencapsulation is being
used to protect and control the release of large hydrophobic or
hydrophilic agents, such as siRNA. The first line of protection is
achieved by loading the siRNA into primary nanocarriers (.about.100
nm), while the second line of stability is obtained by
encapsulating the primary nanocarriers into sub-micron
nanoparticles, typically with a polyethylene glycol (PEG) moiety
anchored to their surface. The nanoparticles formation (typically
nanocapsules, or NCs) is carried out using a nanospray drying
technique [16,23,24].
[0215] The following two types of nanoparticles are herein
described:
[0216] (a) PLGA (Poly D,L-lactic-co-glycolic acid) NPs loaded in
nanocapsules were prepared using hydrophilic coating polymers;
and
[0217] (b) HSA (Human serum albumin) NPs loaded in nanocapsules
were prepared using hydrophobic coating polymers.
[0218] In both cases, a cationic lipid, DOTAP
(1,2-dioleoyl-3-trimethylammonium-propane), was added to the
primary NPs to effectively load the negatively charged siRNA and to
further facilitate `endosomal escape` of siRNA after NPs cell
internalization. Since all ingredients are FDA approved, such a
delivery system provides a platform for systemic delivery of
hydrophilic bio-macromolecules (such as siRNA) improving the drug's
half-life, biodistribution and pharmacokinetics.
[0219] In drug delivery, nanoparticles (NPs) are favored over
microparticles not only due to their ability to enhance drug
efficacy, but to alter favorably the pharmacokinetic profile of the
selected drug since they can be intravenously administrated. In
addition, such nanosized systems are superior to microparticles in
their penetration properties and targeting to specific cell types
[12]. The targeting efficiency and prolonged circulation time of
NPs are the two most important factors for their successful
applications to drug delivery [13]. Targeting NPs can be active
(through attachment of ligands specific for receptors at the cell
target site, or passive. In passive targeting, the tendency of
small NPs to accumulate in the solid tumor due to the phenomenon
known as `Enhanced Permeation and Retention` (EPR) effect is
utilized [14]. Cellular uptake of NPs was found to be dependent on
NP's size, geometry, charge and cell type. In general, particles
smaller than 1 .mu.m can be internalized into cells through several
endocytotic pathways [13]. Furthermore, attachment of polyethylene
glycol (PEG) moieties to the surface of NPs results in steric
hindrance that leads to reduced aggregation and plasma protein
adsorption (opsonization) as well as uptake by the
reticuloendothelial system (RES)--while prolonging blood
circulation time [15]. Finally, to protect from aqueous phase
degradation and ensure stability of NPs upon long term storage, dry
powdered formulations are required. Lyophilization (freeze drying),
usually accompanied with the addition of cryoprotecting excipient
or spray drying processes are the two major, well established
procedures applied for such a purpose.
[0220] Spray drying is a process which converts liquids or
suspensions into dry powders at a continuous single step process.
However, this technique fails to efficiently form and collect fine
particles <2 .mu.m [16]. Recently, a new generation of
laboratory scale spray dryers was developed by Buchi, enabling the
generation of particles in the size range of 300 nm to 5 .mu.m for
small samples quantities (few milligrams or milliliters) at high
yields (>70%). This technology allowed the formation of NPs by
spray drying, resulting in the general structure shown in FIG. 1A
which is a nanoparticle comprising a plurality of nanocarries, the
nanocarriers comprising an active agent. This nanospray dryer
(NSD), schematically illustrated in FIG. 1B, utilizes a vibrating
mesh technology for fine droplets generation. Generally describing,
a piezoelectric crystal driven spray head is incorporated with a
small spray cap that contains a thin perforated membrane (spray
mesh) having an array of precise micron-sized holes, which upon
vibration, creates millions of sized droplets in range of 3-15
.mu.m (typical median size of 5-7 .mu.m, depending on the mesh
size). Furthermore, unlike conventional spray dryers operating on
turbulent flow, this new technology operates on a laminar flow;
hence gentle heating is achievable, thus making the system
compatible for heat-sensitive biopharmaceutical products.
[0221] Over the last decade, in addition to small molecular drugs,
bio-macromolecules delivery, such siRNA is considered for therapy,
using NPs/NCs as carriers. siRNA (small interfering RNA), a short
sequence of RNA molecules (19-30 bp long duplexes) can be used to
silence the expression of a specific gene, via inducing degradation
on its complementary mRNA, in a well defined mechanism [17]. Since
the discovery of siRNA, numerous attempts were made to develop
drugs based on siRNA. However, major barriers arise in delivery of
siRNA, due to its physicochemical nature. siRNA is a large
(.about.13 kDa), hydrophilic, negatively charged molecule and as
such requires transfection vehicles to penetrate the cell membrane
and to gain access to the cytosol. Furthermore, after cell
penetration (usually by endocytosis), an `endosomal escape`
mechanism is required. In addition, systemic delivery of free siRNA
is hampered due to very short half-lives in the blood and fast
renal clearance. To overcome these disadvantages in delivery of
siRNA in-vivo, a variety of chemical modifications were introduced
on the siRNA molecule, preserving its activity and thereby
improving its resistance to RNAses cleavage and increasing its
half-life in human serum [18]. In addition, naked or chemically
modified siRNAs were incorporated in diverse delivery systems,
based on non viral lipids (cholesterol, liposomes), protein
carriers (fusogenic or cell-penetrating peptides), cyclodextrin or
biodegradable polylactide copolymers nanoparticles with or without
conjugation with cationic lipids.
[0222] The nanocarriers of the invention, without wishing to be
bound by theory, provide protection, biocompatibility, improved
stability, desired biodistribution and pharmacokinetics profiles to
the encapsulated hydrophilic bio-macromolecule (i.e. siRNA),
resulting in a unique delivery system with improved therapeutic
properties.
Materials and Methods
Materials
[0223] PLGA: Poly (D,L-lactic-co-glycolic acid) (50:50) (R504H) MW
48,000 Da, and PEG-PLGA (RGP50105) MW 5,000+45,000 Da were
purchased from Boehringer (Ingelheim, Germany). The following
materials were purchased form the following companies: Dextran 40
(MW 40,000 Da), Teva (Jerusalem, Israel); Sodium Hyluronate (HA),
MW 200,000 Da, Bioberica (Barcelona, Spain); DOTAP
(1,2-dioleoyl-3-trimethylammonium-propane-chloride salt), MW 698.5
Da, Lipoid GmbH (Frigenstr, Germany); Commercial Human Serum
Albumin (HSA) 20% solution for i.v. injection (Zenlab 20 or
Biotest), Kamada (Beit-Kama, Israel) and supplied by Hadassah
hospital. HAS, MW 66,500 Da, Macrogol 15 hydroxystearate (Solutol
HS 15) obtained from BASF (Ludwigshafen, Germany). Polyethylene
glycol (PEG) MW 4,000 Da, Polysorbate 80 (Tween 80), Gluteraldehyde
8% sol. in water, Trypsin (from porcine pancreas), RNase &
DNase free Ultra pure water and Phosphate buffered saline (PBS)
(Bioreagent, pH 7.4), were all purchased from Sigma (St. Louis,
Mo., USA). Acetone, Ethanol, Dichloromethane, Chloroform, and
Acetonitrile were all HPLC grade. Other chemicals and solvents were
of analytical reagent grade and used without further purification.
For all experiments done with siRNA only Ultra pure water was used
(Sigma or Beit-Haemek), while for all the other blank systems
(without siRNA), double-distilled water (DDW) was used throughout
the study.
[0224] siRNA:
[0225] Anti EGFR (Epidermal Growth Factor Receptor) siRNA
(EGFR-siRNA) (21 bp, MW 13,400 Da) and scrambled siRNA (21 bp, MW
13,821 Da), for control purpose were purchased from Ambion (Austin,
Tex., USA).
TABLE-US-00001 EGFR-siRNA: (SEQ ID NO: 1) S (5'.fwdarw.3')
CCAUAAAUGCUACGAAUAUtt (SEQ ID NO: 2) AS (5'.fwdarw.3')
AUAUUCGUAGCAUUUAUGGag scrambled siRNA: (SEQ ID NO: 3) S
(5'.fwdarw.3') UAACGACGCGACGACGUAATT (SEQ ID NO: 4) AS
(5'.fwdarw.3') UUACGUCGUCGCGUCGUUATT
[0226] In the above sequences, chemical modifications consisted of
a few LNA modifications; lower case letter=DNA base.
[0227] Anti Green Fluorescent Protein (GFP) siRNA (GFP-siRNA) (21
bp, MW 14,352 Da), and Cholesterol modified GFP-siRNA (Chol-GFP
siRNA) (21 bp, MW 15,079 Da), were provided by Roche (Kulmbach,
GmbH) and were used for most of experiments (especially for
estimation of drug loading).
TABLE-US-00002 GFP-siRNA: (SEQ ID NO: 5) S (5'.fwdarw.3')
AuAucAuGGccGAcAAGcAdTsdT (SEQ ID NO: 6) AS (5'.fwdarw.3')
UGCUUGUCGGCcAUGAuAUdTsdT Chol-GFP siRNA: (SEQ ID NO: 7) S
(5'.fwdarw.3') (Chol)-linker-AuAucAuGGccGAcAAGcAdTsdT (SEQ ID NO:
8) AS (5'.fwdarw.3') UGCUUGUCGGCcAUGAuAUdTsdT
[0228] In the above sequences, chemical modifications: lower case
letter=2' O-methylated nucleoside, dT=desoxy-thyamin,
sdT=desoxy-thyamin phosphorothioate, underlined=overhang.
TABLE-US-00003 Anti-EGFR-siRNA synthesized 'in house' using
EGFR-siRNA (SEQ ID NO: 9) S* (5'.fwdarw.3') CCAUAAAUGCUACGAAUAUtt
(SEQ ID NO: 10) AS (5'.fwdarw.3') AUAUUCGUAGCAUUUAUGGtt and (SEQ ID
NO: 11) S* (5'.fwdarw.3') CCAUAAAUGCUACGAAUAUtt (SEQ ID NO: 12) AS
(5'.fwdarw.3') AUAUUCGUAGCAUUUAUGGtt
[0229] In the above sequences, chemical modifications:
underlined=2' O-methylated nucleoside, lower case letter=DNA base,
*addition of (Chol)-linker or NIR dye molecule in the 5' position
will also be made.
Methods and Experimental Methodology
PLGA NSs Preparation
[0230] Preparation of nanospheres was made based on the `polymer
interfacial deposition` method [19]. In brief, the polymer PLGA 48
kDa was dissolved in a water-miscible organic solvent (acetone).
Then the organic phase was added rapidly and under stirring
(.about.900 RPM) to the aqueous phase, which typically contains a
surfactant (Solutol.RTM. HS 15). As a fast escape of acetone to the
water phase occurs, the hydrophobic polymer spontaneously forms
spherical negatively charged nanometric spherical particles (50-200
nm).
[0231] For formation of positively charged PLGA NSs, DOTAP
(1,2-dioleoyl-3-trimethylammonium-propane-chloride salt) was added
to the acetonic phase in different percentages. The formulations
were evaporated (at 37.degree. C.) to remove all acetone traces,
and further concentrated to a final volume of 5 ml aqueous phase,
then centrifuged for 10 min at 4,000 RPM (precipitation was removed
and dried--found to be no more than 1% (w/w)), and washed
(.times.10) with DDW using VivaSpin-6 (300 kDa, Vivascience) in
order to reduce the percentage of Tween. The final percentage of
each component in the washed formulation was: 1.5% (w/v) PLGA and
0%, 0.04%, 0.2% or 0.4% (w/v) DOTAP (Formulations A, B, D and C,
respectively).
Size, Size Distribution and Zeta Potential Measurements of Primary
NSs
[0232] Physicochemical characterization of primary NSs (made from
PLGA or from crosslinked HSA) was measured by dynamic light
scattering, using Zetasizer Nano ZS (Malvern Instruments, Malvern,
UK). All samples were diluted 1:100 in HPLC grade water (pH=5.5)
prior to measurements. When siRNA loaded NSs were characterized,
RNase free water with 0.01% NaCl was used (pH=5.5).
Loading Efficacy of siRNA in PLGA NSs
[0233] For incubation with siRNA, the formulation were made at the
same manner detailed above, but instead of using water (HPLC grade)
as aqueous phase, RNase free water (RNFW) were used for formation
and washing steps. Briefly, 0.1 ml of washed PLGA NSs were taken
from each formulation (1.5 mg PLGA content) and incubated with
different amounts of GFP-siRNA (50 .mu.g, 100 .mu.g) for lhr with
mild shaking at room temp. After incubation each formulation was
washed (.times.10) with RNFW (using 300 kDa Nanosep centrifugal
centricones, Pall) and the total ultrafiltrate was collected,
lyophilized and reconstituted with 500 .mu.l RNFW, from which, 150
.mu.l were injected to HPLC. GFP-siRNA content in the ultrafiltrate
was calculated based on calibration curves made by reversed phase
HPLC (RP-HPLC).
High-Pressure Liquid Chromatography (HPLC)
[0234] Calibration curves for the different siRNAs used were made
using HPLC (Shimadzu LC-2010C) with a Clarity 3 um Oligo-RP column
50.times.4.6 mm (Phenomenex, USA). The siRNA was dissolved in RNFW
or in RNF buffer (100 mM NaCl, 50 Mm Tris), prior to injection.
Mobile phase: A-- RNase free Buffer (100 mM TEAA), B--Acetonitrile.
Long Gradient (for Cholesterol modified siRNA): B/A (10:90) to
(90:10) in 40 min, then another 10 min of B/A (10:90). Short
Gradient (for none Cholesterol modified siRNA): B/A (10:90) to
(40:60) in 15 min, then another 10 min of B/A (10:90). Flow rate: 1
ml/min, UV-- Detection: 260 nm and 280 nm.
Gel Electrophoresis with Polyacrylamide Gel (PAGE)
[0235] For siRNA integrity (stability) evaluation 8% (19:1) native
polyacrylamide gels (PAGE) was used. Electrophoresis was carried
out at 200V for 50 min, in Tris-Borate-EDTE (TBE) as running
buffer. For siRNA staining, 0.01% of EtBr was used. Gels were
visualized under a UV transilluminator.
Protocol for Evaluation of Loading Efficacy of GFP-siRNA on PLGA
NSs
[0236] PLGA NSs are dissolved in chloroform until a clear solution
forms, followed by the addition of an equal volume of RNFW. Samples
are vortexed and centrifuged (5 min at 4,000 RPM). The upper
aqueous phase (with free siRNA) is collected. This procedure is
repeated twice. Next, the collected aqueous phase is lyophilized.
Determination of siRNA content is assessed by RP-HPLC, after
reconstitution of lyophilized samples in RNF buffer. Since NSs with
DOTAP and siRNA may form an `ion-pair`, addition of heparin (highly
negative charged molecule) to aqueous phase is examined, following
an incubation for 1 hr at 37.degree. C., under mild shaking, in
order to ensure that all siRNA is released in its free form.
Primary Crosslinked HSA NSs Preparation
[0237] Prior to use, commercial HSA was desalted for 24 hr in DDW
using a cellulose membrane (MWCO 14,000) from Medicell
International (Liverpool road, London), in order to remove salts
and all preservative traces. In order to produce nanometric HSA
NPs, the well known method of pH-coacervation [20] (also known as
desolvation technique) was applied. Briefly, HSA solution, adjusted
to a specific pH, was transformed into nanospheres by continuous
(.about.1 ml/min) addition of desolvating agent, under constant,
rapid stirring (.about.960 RPM or 40 HZ) at room temp. Addition of
desolvating agent was continued until sufficient turbidity appeared
(usually between 40 to 80% (v/v) of desolvating agent), then the
crosslinking process was carried out with glutaraldehyde for at
least 2 hr at room temperature under milled shaking. After
crosslinking, desolvating agent was evaporated (37.degree. C.) and
centrifuged for 10 min at 4,000 RPM (precipitation was removed,
dried and determined gravimetrically). NS s were washed (.times.10)
with DDW, at three different cycles of centrifugation (4,000 RPM,
4.degree. C.), using vivaspin 300 kDa (Viva science). In some cases
acetone phase contained DOTAP.
Loading Efficacy of siRNA on Crosslinked HSA NSs
[0238] For encapsulation of siRNA inside crosslinked HSA NSs, siRNA
was added to HSA solution prior the addition of desolvating phase.
The rest of procedure was made exactly in the same manner detailed
above, when instead of using DDW, RNAse free water (RNFW) was used.
After crosslinking, the NSs were washed and the total ultrafiltrate
was collected, lyophilized and reconstituted with 500 .mu.l RNFW,
from which, 160 ul were injected to HPLC. Since partial degradation
of free siRNA in the ultrafiltrate was observed, the preferred way
to determine siRNA content in the NS s is to directly determine
siRNA content in the particle (after its digestion), and not based
on the free siRNA (un-encapsulated) in the ultrafiltrate.
A Protocol for Determination of siRNA Content in the Crosslinked
HSA NSs
[0239] The total weight of HSA per 100 .mu.l of suspension (after
wash) was quantified gravimetrically. Then 1-2 mg of washed HSA NPs
encapsulating siRNA was diluted to 1 ml with RNase free PBS buffer
(adjusted to pH=7.5 using 0.5 M NaOH solution) and digested with 20
to 150 .mu.g Trypsin for 60, 90 or 180 min, at dark and under mild
shaking at 37.degree. C., till a clear solution was formed. In case
sample containing DOTAP, Heparin (90 .mu.g) was added to the
aqueous phase, 0 or 60 min after the addition of Trypsin. The free
siRNA quantity was determined using RP-HPLC, in the presence of
Tryp sin or without it.
[0240] The Trypsin as well as the fragments of digested HSA can be
removed by precipitation using (phenol/chloroform) (1:1)
mixture.
Encapsulation of PLGA NSs into NCs Using Nanospray Dryer--Aqueous
Mode
[0241] NCs were prepared via spray drying on the Nano Spray Dryer
B-90 (Buchi Labortechnik AG, Flawil, Switzerland), operating at
`open loop` mode, hence air was flowing through the system. In all
experiments gas flow was about 120 l/min. 100% spraying and 4 .mu.m
mesh size membrane were used in all experiments.
Encapsulation of HSA NSs into NCs using nanospray dryer--Organic
mode
[0242] NCs were prepared via spray drying on the NSD B-90 operates
at `closed loop` mode, hence, N.sub.2 (g) and CO.sub.2 (g) are
flowed in the system instead of air. In all experiments gas flow
was about 120 l/min. The air soaked with volatile vapors and
humidity, transferred to a
[0243] Dehumidifier unit, for drying and condensation, then
returned dry to the system in a circular path. Spray drying was
carried out at low temperatures (Tin=30.degree.-60.degree. C.) with
mesh size membrane 4 .mu.m.
Melting Point Measurements for siRNAs
[0244] Melting point measurements for different siRNAs (21-mer)
used were performed on a UV-visible spectrophotometer (Cary 300) at
260 nm, by elevating the sample temperature from 20.degree. C. to
85.degree. C. at rate of 1.degree. C./min. All siRNAs were
dissolved in buffer (48 mM Tris, 96 mM NaCl, pH 7.1) to obtain a
concentration of 4-10 ng/.mu.l.
Thermal Analysis by Differential Scanning Calorimeter (DSC)
[0245] DSC measurements were made for the polymers (PLGA 48 kDa and
PEG-PLGA 50 kDa), and performed at a temperature range of
-20.degree. C. to 220.degree. C. using a Mettler DSC 1 Star System
(calibrated with In standards) at a heating rate of 10.degree.
C./min, under a nitrogen atmosphere.
SEM (Scanning Electron Microscope) and EDS (Energy Dispersive X-Ray
Spectroscopy)
[0246] Geometry, size and surface morphology of the spray-dried NCs
(and encapsulated NSs) were observed by a High Resolution Scanning
Electron Microscope (HR-SEM) with High stable Schottky Field
Emission Source (Sirion, model: Quanta 200 FEI, Germany), 5 kV.
Prior to imaging, the samples were dispersed onto carbon sticky
tabs and coated with gold and palladium mixture for 90-120 sec. In
case of primary NSs dispersed in water, the samples were highly
diluted, then spattered on glass and left to evaporate overnight.
Element analysis of the specimen was made by EDS (Energy Dispersive
X-ray Spectroscopy), with X-MAX20 SDD Inca 450 EDS LN2 free
detector (Oxford Instruments, UK), using low voltage of 5 kV, with
spectral resolution of 129 eV.
A Method for Evaluation of Size Distribution for Spray Dried
NCs
[0247] Since the regular Zetasizer Nano ZS is limited for
measurements of particles smaller than 4 .mu.m as well as for
relatively homogeneous dispersions, a sufficient particle size
distribution for the spray dried NCs can only be made by means of
laser diffractometry using a Mastersizer 2000E (Malvern
Instruments, UK). Approximately 4 mg of sample was needed for each
measurement in order do disperse it at 120 ml of dispersant.
[0248] Span value was calculated by:
Span=(d90-d10)/d50
[0249] wherein d50 was the volume median size; d90, 90% of the
volume had a size smaller than d90; d10, 10% of the volume had a
size smaller than d10.
[0250] Low span value indicated a narrow size distribution.
A Protocol for Determination of the Spray Drying Encapsulation
Efficacy
[0251] For separation between different NCs populations by size,
size exclusion chromatography (SEC) was applied. Then, in order to
determine the content of HSA NSs encapsulated inside a specific
population of large PLGA NCs, first desolvation of the PLGA NCs in
chloroform was made. Then, upon centrifugation (10,000 RPM, 15 min)
the primal HSA NPs was separated as sediment, isolated and its
content was validated by Bicinchoninic Acid (BCA) Protein Assay kit
or by the nitrogen content (detected by simple microanalysis). For
HSA NCs encapsulating primary PLGA NPs, NCs with known weight was
degraded upon incubation with aqueous solution of Trypsin (PBS
buffer pH of 7.5 at 37.degree. C.), then HSA content was quantified
using BCA and the PLGA quantity was estimated by subtraction. When
NCs encapsulating primary NSs loaded with siRNA, the total content
of isolated siRNA was determined (after NCs disassembling and NSs
digestion).
In-Vitro Release Kinetic Profile Determination of EGFR-siRNA
[0252] The kinetic profile for EGFR-siRNA released from primary NSs
and secondary NCs, will be determinate in-vitro, in the same manner
detailed at Hagigit et al. [21]
Cell Culture
[0253] A-431 human epithelial squamous carcinoma cells and other
colorectal carcinoma cells, will be maintained in Dulbecco's
Modified Eagle's Medium (DMEM) containing 10% fetal bovine serum, 2
mM L-glutamine and 10000 U/ml penicillin and 100 ug/ml
streptomycin. The medium will be replaced every two days. The cells
will be grown at 37.degree. C., 6% CO.sub.2 in a CO.sub.2
incubator. Confluent flasks will be splitted at 1:10 ratio after
trypsinization of the cultures with 0.25 ml Trypsin solution (Beit
Haemek, Afula, Israel). All experiments will be carried out in a
clean room according to ISO7 requirements (10000
particles/m.sup.3).
NPs Stability in Cell Culture Medium
[0254] Size measurements, using Zetasizer Nano ZS will be made for
the NSs, after different times of incubation in A-431 cell culture
medium, in the same manner as detailed at [22].
NCs Stability Evaluation
[0255] Size measurements using Mastersizer X, and morphology
evaluation using SEM, will be made for the final NCs after storage
in different conditions (4 and 37.degree. C.) for 4, 8 and 12
weeks.
NPs Cytotoxicity in A-431 Cells
[0256] The cell proliferation will be tested over a time period of
144 h, in the presence of 5, 1, 0.1 and 0.01 mg/ml NPs
concentration per well, in the same manner described at [21].
Uptake of FITC-Labeled HSA NSs in A-431 Cells
[0257] In order to produce a 2% FITC-labeled HSA NSs, 1.9 mg of
FITC-BSA (Bovine Serum Albumin) was added to 4 ml of 2% HSA
solution. The particles were made in the same way as detailed
previously, just under dark. The washed NSs were then filtrated
through cellulose acetate sulphonate filter (0.2 um, Whitman).
Next, aliquots of the FITC-labeled HSA NPs (61.2 .mu.l and 122.45
.mu.l) were diluted in DMEM buffer into a total volume of 1.5 ml,
in order to produce concentrations of 1 mg/ml and 2 mg/ml,
respectively, per well. For cell labeling, 150,000 A-431 cells were
placed on cover slides and left overnight to adhere. The following
day the adherent cells were incubated with the aliquots of
FITC-labeled HSA NSs for 4 h or 22 h, followed by three washes with
phosphate buffered saline (PBS). Thereafter the cells were fixed
with 4% paraformaldehyde (Sigma-Aldrich) and washed three times
with PBS. In the negative control experiments, the FITC-labeled HSA
NSs incubation step was omitted while the other steps remained the
same. The cells were examined in a FluoView FV300 confocal laser
scanning microscope (Olympus, Tokyo, Japan).
Evaluation of Anti EGFR-siRNA Silencing Efficacy in A-431 Cells
Using an in-Cell NIR Model
[0258] Anti EGFR-siRNA silencing efficacy will be performed based
on the novel method developed and detailed in Cohen et. al [23].
Knockdown efficacy of EGFR mRNA will be confirmed by RT-PCR
(Reverse Transcription--Polymerase Chain Reaction) using relevant
primers and EGFR protein levels will be quantified by western
blotting.
Xenograft Tumor Studies in Mice
[0259] A-431 tumor cells will be cultured. Subconfluent cells
(70%-80%) will be harvested after brief treatment with 0.25%
trypsin and resuspended in Hank's balanced salt solution for
inoculation. Tumor cell suspensions (3-5.times.10.sup.6 cells) will
be injected SC in a volume of 0.2 ml into the right flank of each
mouse. 8 to 10 mice will be randomly assigned to each treatment
group, and treatments will be conducted for up to 4 weeks. The
doubled nanovehicle will be injected in the jugular vein, at the
appropriate dosage. Tumor measurements will be made periodically
with manual calipers (at least once a week), and tumor volumes will
be calculated using the formula: 0.52.times.length.times.width. At
the end of the study, tumors will be excised and weighed, and then
for some studies. In parallel the tumors will be bioimaged
noninvasively with NIR labeled EGF probe.
Results
PLGA NSs Preparation in Organic Phase
[0260] The first type of primary NSs were prepared from PLGA 48 kDa
(Poly D,L-lactic-co-glycolic acid, 50:50), with or without addition
of cationic lipid (DOTAP), producing negatively or positively
charged NSs. The NSs were prepared using the well established
technology of `polymer-interfacial deposition` method. Four
different blank formulations were selected in order to test siRNA
loading efficacy through interfacial interactions (electrostatic
& hydrophobic). The four formulations (see Table 1), differ in
their DOTAP content (Formulations A, B, D and C, containing 0%,
0.04%, 0.2%, and 0.4% (w/v) of DOTAP, respectively).
Physicochemical Characterization
[0261] Physicochemical characterization for the PLGA NSs was made,
prior and after incubation (1 hr at room temp.) with GFP-siRNA (50
and 100 .mu.g). The results are detailed in Table 1.
[0262] Based on these results, GFP-siRNA adsorption is clearly
observed, leading to a change in the physicochemical nature of the
NSs (ZP, PDI and size). While ZP for negatively charged NPs
(formulation A) remained negative, formulation B changes from the
slightly positive to negative even with 50 .mu.g of GFP-siRNA. For
NSs with a pronounced positive charge (formulations D and C-- ZP
above 40 mV) we observe a decrease in ZP; particularly for
formulation D.
TABLE-US-00004 TABLE 1 Physicochemical characterization for the
PLGA NSs, prior and after incubation (1 hr at room temp.) with
GFP-siRNA Mean diameter Mean ZP Formulation Conditions of
measurement [nm] [mV] PDI A No incubation with siRNA 68.8 .+-. 22.1
-28.6 .+-. 14.8 0.10 After incubation with 50 .mu.g GFP-siRNA and
78.1 .+-. 39.5 -25.8 .+-. 10.7 0.21 washing After incubation with
100 .mu.g GFP-siRNA and 88.9 .+-. 44.5 -27.8 .+-. 10.7 0.17 washing
B No incubation with siRNA 103.1 .+-. 32.7 +11.2 .+-. 9.9 0.09
After incubation with 50 .mu.g GFP-siRNA and 124.0 .+-. 56.7 +44.0
.+-. 7.0 0.12 washing After incubation with 100 .mu.g GFP-siRNA and
226.7 .+-. 98.5 +2.2 .+-. 4.0 0.34 washing C No incubation with
siRNA 119.9 .+-. 46.7 +49.7 .+-. 10.6 0.16 After incubation with 50
.mu.g GFP-siRNA and 145.5 .+-. 62.0 +45.8 .+-. 7.0 0.16 washing
After incubation with 100 .mu.g GFP-siRNA and 204.7 .+-. 114.2
+44.4 .+-. 7.4 0.22 washing
[0263] Polydispersity of size distribution (PDI), mean hydrodynamic
diameter and Zeta potential (ZP), N=3, of negatively or positively
charged primary PLGA NSs before and after incubation with
GFP-siRNA. Incubation was done in duplicates.
Effective Loading of siRNA on Primary PLGA NSs
[0264] In order to determine the loading efficacy of siRNA after
incubation with the different PLGA NSs, NSs were washed and the
content of unbound siRNA in the collected ultrafiltrate was
quantified by RP-HPLC (Table 2). The percent of siRNA associated
with the NSs was calculated from the difference of the total siRNA
(used for incubation), to that of free siRNA in the
ultrafiltrate.
[0265] According to Table 2, in formulations containing 0.2% (w/v)
of DOTAP and above (formulations C and D) full siRNA adsorption
occurs. These results demonstrate that positive Zeta potential of
.about.50 on the PLGA NSs surfaces, induces strong and efficient
adsorption of 100 ng siRNA per 1.5 mg of NSs. Insufficient loading
occurs with slightly positive NSs (formulation B) and intermediate
values occur for negatively charged NSs (formulation A). These
results are further validated by gel retardation assay (PAGE 8%)
(FIG. 2) showing no free siRNA is found in the ultrafiltrate of
formulations C and D (on the contrary to the free siRNA detected in
the ultrafiltrate of formulations A and B).
TABLE-US-00005 TABLE 2 Loading efficacy of GFP-siRNA on 1.5 mg of
PLGA NSs. (*Based on calibration curves, the minimum amount of
siRNA needed for detection through RP-HPLC is 360 ng (0.7% of 50
.mu.g), hence, the percent of siRNA associated can be determined
only in 99.3% accuracy. ND--not detected.) Quantity of GFP-
Calculated average of free GFP- Calculated average of GFP- siRNA
used for siRNA at total ultrafiltrate siRNA adsorbed to PLGA NPs
Formulation incubation [.mu.g] Quantity [.mu.g] Percentage [%]
Quantity [.mu.g] Percentage [%] A 50 16.4 .+-. 0.8 32.7 .+-. 4.7
33.7 .+-. 1.6 67.3 .+-. 4.7 100 56.6 .+-. 2.8 56.6 .+-. 4.9 43.4
.+-. 2.1 43.4 .+-. 4.9 B 50 28.8 .+-. 3.8 57.5 .+-. 13.2 21.3 .+-.
2.8 42.5 .+-. 13.2 100 79.8 .+-. 3.0 79.8 .+-. 3.7 20.2 .+-. 0.8
20.2 .+-. 3.7 C 50 ND ND *50 *Above 99 100 ND ND *100 *Above 99 D
50 ND ND *50 *Above 99 100 ND ND *100 *Above 99
[0266] The observation that formulation B shows poor loading
capacity, can be explained by its low stability and tendency to
aggregate (typical for particles with ZP value smaller than 20 mV).
No fragmentation of the free siRNA was observed in HPLC
chromatograms or upon evaluation by gel retardation assay (PAGE
8%), see FIG. 2.
HSA NSs Preparation in Aqueous Phase
[0267] Primary NSs .about.100 nm, made from crosslinked HSA (Human
Serum Albumin) were examined. The method of pH-coacervation was
applied for this purpose. By changing different parameters (pH,
type and quantity of desolvating agent used (ethanol or acetone), %
HSA in solution and stirring speed) control the size of the formed
NSs was achieved. The best results were obtained at 2% HSA
solutions when acetone was used as desolvating agent. Selected
results are shown in Table 3 and indicate that the smallest
particles were obtained with acetone at pH 7 and 9.
TABLE-US-00006 TABLE 3 Physicochemical characterization for the HSA
NPs (*Massive sediment appeared (64% (w/w)). In other samples
sediment was negligible). Total Mean Desolvating acetone diameter
Mean ZP Sample pH agent added [ml] [nm] [mV] PDI 1* 7 Ethanol 10
241 .+-. 71 -35 .+-. 5 0.07 2 8 Ethanol 10 166 .+-. 44 -35 .+-. 8
0.05 3 9 Ethanol 7 152 .+-. 43 -51 .+-. 8 0.06 4 7 Acetone 10 58
.+-. 30 -38 .+-. 9 0.24 5 8 Acetone 21 131 .+-. 55 -36 .+-. 7 0.14
6 9 Acetone 9 61 .+-. 25 -54 .+-. 10 0.13 (53%) -37 .+-. 8
(25%)
Physicochemical Characterization
[0268] The physicochemical characterization for the crosslinked HSA
NSs, shows that small, spherical and negatively charged NSs were
formed, and their size distribution (from 40 to 300 nm) and
polydispersity, are influenced by the pH and type of desolvating
agent (Table 3 and FIGS. 3A-3B). In all pH tested, acetone produced
smaller NSs with higher PDI values compared to ethanol.
[0269] Polydispersity of size distribution (PDI), mean hydrodynamic
diameter and Zeta potential (ZP), N=3, of primary crosslinked HSA
NSs prepared from 4 ml (2% HSA solution) at different pH with
acetone or ethanol as desolvating agent, stirred at -960 RPM.
[0270] The isoelectric point of HSA is about 5.0. As the pH of the
aqueous solution is more basic, we gain more negatively charged
carboxylic group on its surface that repels HSA molecules. Based on
this idea, the smaller particles (.about.100 nm) are formed at pH
9. However, by utilizing the process described above, HSA NSs at
the required size were obtained already at pH 7, by using acetone
instead of ethanol (Table 3).
Effective Loading of siRNA in Primary HSA NSs
[0271] To encapsulate siRNA effectively inside the crosslinked HSA
NSs, a scaling down process was made, requiring additional
adjustment of parameters; working with 0.6 ml of 2% HSA solution
instead of 4 ml. In addition, the cationic lipid DOTAP
(`endocytosic agent`) was also added to increase siRNA
encapsulation efficacies. Blank systems (without siRNA) displayed
in Table 4, showed that in some cases massive sediment appeared
together with the formation of NSs. The best results obtained at pH
8 and 9 (samples 5, 9 and 10)--small NSs formed with the lowest
sediment percentage.
TABLE-US-00007 TABLE 4 loading of siRNA in primary HAS NSs Total
acetone Mean Mean ZP Sediment Sample pH DOTAP added [ml] diameter
[nm] [mV] PDI % [w/w] 1 6.6 - 1.5 155 .+-. 55 -36 .+-. 9 (78%) 0.12
90 0.7 .+-. 4 (18%) 2 6.6 + 0.5 276 .+-. 156 -44 .+-. 5 0.26 81 3
7.3 - 1.5 139 .+-. 43 -41 .+-. 8 0.08 54 4 7.3 + 1.5 129 .+-. 55
-43 .+-. 8 0.16 60 5 8 - 1.5 80 .+-. 28 -43 .+-. 10 0.13 21 6 8 +
1.5 90 .+-. 36 -48 .+-. 11 0.13 43 7 8 - 2.4 228 .+-. 143 -19 .+-.
5 0.24 <1 8 8 + 2.4 3587 .+-. 330 -7 .+-. 3 0.29 <1 9 9 - 1.5
135 .+-. 62 -63 .+-. 7 (50%) 0.26 1 -36 .+-. 9 (50%) 10 9 + 1.5 79
.+-. 43 -28 .+-. 6 0.26 11
[0272] Based on these findings, encapsulation of 200 .mu.g siRNA
was also performed in basic pH (Table 5).
TABLE-US-00008 TABLE 5 loading of siRNA in primary HAS NSs, 200
.mu.g siRNA Total Mean siRNA acetone diameter Mean ZP Sediment
Sample pH DOTAP type added [ml] [nm] [mV] PDI % [w/w] 1 9 - GFP 1.5
107 .+-. 31 -52 .+-. 6 (63%) 0.07 8 -31 .+-. 8 (37%) 2 9 - Chol-GFP
1.5 109 .+-. 39 -34 .+-. 8 (68%) 0.13 15 -62 .+-. 5 (32%) 3 9 + GFP
1.5 88 .+-. 30 -33 .+-. 9 (90%) 0.1 33 -28 .+-. 5 (10%) 4 9 +
Chol-GFP 1.5 113 .+-. 32 -42 .+-. 9 0.07 <1 5 8 + GFP 1.5 81
.+-. 32 -35 .+-. 8 0.23 <1 6 8 + Chol-GFP 1.5 119 .+-. 53 -37
.+-. 7 0.34 <1 7 8 + GFP 2.4 171 .+-. 90 -23 .+-. 5 0.32 <1 8
8 + Chol-GFP 2.4 88 .+-. 33 -19 .+-. 6 (40%) 0.14 <1 -48 .+-. 7
(40%) 9 7 + GFP 1.5 93 .+-. 39 -43 .+-. 6 0.14 0 10 7 + Chol-GFP
1.5 93 .+-. 39 -23 .+-. 8 (70%) 0.13 0 -39 .+-. 6 (30%)
[0273] Addition of desolvating agent in high volumes (70-80% (v/v))
was made to promote HSA NSs formation, but also to induce a
sufficient siRNA encapsulation. Another strategy to obtain high
encapsulation, was to include the lipophilic derivate of GFP-siRNA
(5'-Cholesteryl-GFP-siRNA, i.e. Chol-GFP-siRNA). The results
detailed in Table 5 indicate that at pH 8 and 7, with samples
containing DOTAP, addition of siRNA (GFP or Chol-GFP), reduced
dramatically the appearance of sediment (as observed in the blank
samples upon addition of 1.5 ml of acetone--Table 4). At pH 9, the
same phenomenon was seen only when Chol-GFP-siRNA was used.
[0274] Polydispersity of size distribution (PDI), mean hydrodynamic
diameter and Zeta potential (ZP), N=3 and weight percentage of
sediment evolved upon formation of primary crosslinked HSA NSs,
prepared from 0.6 ml (2% HSA solution) at different pH with
different amounts of acetone. (+/-) refers to acetone phase with
0.03 mg DOTAP or without DOTAP (a-acetone phase with 0.015 mg
DOTAP).
[0275] Physicochemical characterization of primary crosslinked HSA
NSs made with acetone upon encapsulation of 200 .mu.g of GFP-siRNA
or Chol-GFP-siRNA. (+/-) refers to acetone phase with 0.03 mg DOTAP
or without DOTAP. Molar ratio of DOTAP: siRNA is 3:1.
[0276] SEM characterization was made to the new crosslinked HSA NSs
(loaded with DOTAP and siRNA) (FIGS. 4A-4B). It is clearly seen
that the addition of DOTAP, as well as the encapsulation of siRNA
(GFP or Chol-GFP), did not change the spherical shape of the formed
NPs, already observed for the blank system (FIG. 3).
[0277] In order to determine the encapsulation efficacy of siRNA in
the crosslinked HSA NSs, the washed NSs were digested with Trypsin
till a clear solution was formed and the total siRNA content was
detected using RP-HPLC and calculated based on AUC. According to
the protocol we have developed in our lab (for detailing see
section E), the reliability of such method in quantification the
released siRNA, is highly efficient (FIGS. 5-6). The encapsulation
efficacies for the HSA NSs produced in different pH conditions are
summarized in Table 6. A few trends can be concluded; In the
presence of DOTAP, the best encapsulation (.about.40%) accepted at
the low basic pH (pH 8 and 7). At pH 9, removal of the DOTAP from
NSs, have reduced encapsulation efficacies. This effect is more
pronounced when the non cholesterol modified siRNA (GFP-siRNA) is
used.
TABLE-US-00009 TABLE 6 encapsulation efficacies for the HSA NSs,
200 .mu.g of siRNA - Encapsulation efficacy summary for crosslinked
HSA NSs made with 200 .mu.g siRNA (GFP or Chol-GFP) at different pH
conditions (pH of 7, 8 or 9) with or without addition of 0.03 mg
DOTAP. Experiment was made in duplicates and the average value is
displayed. Determination was made by RP-HPLC. pH DOTAP siRNA type %
encapsulation Encapsulated isRNA (.mu.g) 9 - GFP 9 18 9 + GFP 24 48
8 + GFP 42 84 7 + GFP 43 86 9 - Chol-GFP 16 32 9 + Chol-GFP 26 52 8
+ Chol-GFP 40 80 7 + Chol-GFP 39 76
Nanoencapsulation of Primary NSs into NCs by Nanospray Drying
Approach
PLGA NS Loaded Nanocapsules Preparation Using Hydrophilic Coating
Polymers
[0278] To produce stable, small, spherical NCs (empty or loaded
with primary PLGA NSs), with intact envelop and in high yields,
numerous formulations with different parameters were
investigated:
[0279] (1) type of water soluble polymers including Dextran 40
(MW=40 kDa), Sodium Hyluronate (HA, MW=200 kDa) and Human serum
Albumin (HSA, MW=66.5 kDa); (2) % (w/v) of hydrophilic polymer and
PLGA NSs in water dispersed phase; (3) T.sub.in (T.sub.1n is the
inlet temperature, the temperature of the drying air/gas which
flows linearly), and (4) addition of surfactant (Tween 80). In
order to form the smallest submicron droplets, the smallest mesh
size membrane (4 .mu.m) was used.
[0280] The overall results of the extensive formulations studied
led to the following observations:
[0281] (1) Spraying process with HA was insufficient due to its
high viscosity (even at 0.1% (w/v) solution).
[0282] (2) PLGA NSs encapsulation using Dextran produced spherical
NCs with perforated surfaces (FIG. 7).
[0283] (3) The best results were obtained when HSA was used at a
range of concentrations of 0.1% to 1.6% (w/v) in aqueous phase.
[0284] (4) Empty HSA NCs were successfully prepared, as well as HSA
NCs containing various amounts of PLGA NSs (positively and
negatively charged).
[0285] (5) SEM measurements confirmed that: (a) In all cases,
small, spherical NCs exhibiting intact surface were formed, and (b)
When the concentration of HSA in DDW decreased, smallest NCs were
formed (FIGS. 8A-8H). From FIGS. 8A-8H, it is deduced that the
decrease in HSA concentration from 1.6% to 0.25% (w/v) resulted in
the formation of NCs with decreasing size. Specifically, a small
fraction of NCs decreased from 7 to 2 .mu.m while most NCs
population decreased from 2 to 0.5 .mu.m, respectively.
[0286] Optimization of nanoencapsulation process Optimization for
the spray drying process was made only for the following
parameters:
[0287] (1) Surface morphology--the best results obtained when HSA
was used, as reported above.
[0288] (2) Particle size--upon reduction of solid concentration in
the aqueous phase, the smallest NCs (0.3-2 .mu.m) are formed, as
reported above.
[0289] (3) Yields--addition of surfactant (Tween 80) at 0.06% (w/v)
led to significant improvement of the yields achieved in the
process, and in some cases improved the spraying rate (Table
7).
TABLE-US-00010 TABLE 7 Optimization for the nanospray drying
process performed at aqueous mode (*with negatively charged PLGA NS
s in a molar ratio of 4:1 HAS:PLGA) Yield [% w/v] Spraying rate
[ml/min] HSA in aqueous Without with 0.06% Without with 0.06%
solution* [% w/v] Tween 80 Tween 80 Tween 80 Tween 80 0.1 -- 96 --
0.05 0.25 11 77 0.02 0.03 0.5 30 98 0.02 0.16 0.75 21 90 0.03
0.05
[0290] Operating temperature--to produce dried NCs, different
temperature of the drying air (T.sub.in) were tested (80, 100 and
120.degree. C.). Attempts to form dried NCs at 70.degree. C. were
not effective. We found that spray draying 80.degree. C. was quite
effective for our formulations, when siRNA was not included.
[0291] The temperature at which the 50% of siRNA strands are
denatured is called the melting temperature, or Tm. The Tm for
various siRNAs (21 mers) used in our research, was measured and
found to be: 78, 77 and 72.degree. C. for GFP-siRNA, Chol-GFP-siRNA
and EGFR-siRNA, respectively. With all examined siRNAs, beginning
of separation was already observed at 60.degree. C. (data not
shown).
[0292] Based on these results, we can conclude that exposing siRNAs
to temperatures higher than 60.degree. C. is not recommended,
leading us to favor nanospray drying process performed at low
temperatures (<60.degree. C.), usually efficient upon working
with volatile organic solvents.
[0293] HSA NS loaded nanocapsules preparation using hydrophobic
coating polymers To produce the desirable spherical submicron NCs
(empty or loaded with primary HSA NSs), with a smooth interface and
in high yields, together with spray drying at low temperatures
(<60.degree. C.) different parameters were changed and tested:
(1) type of organic disperse phase, (2) T.sub.in, (3) type of
hydrophobic polymers used for encapsulation--PLGA 48 kDa and
PEG-PLGA 50 kDa [5 kDa for PEG+45 kDa for PLGA]), (4) % (w/v) of
hydrophobic polymer and crosslinked HSA NSs in organic dispersed
phase, (5) addition of surfactant (Tween 80 or PEG 4000) and (6) %
spraying. Based on our previous experience acquired through
operating the NSD B-90 in aqueous mode, only mesh size membrane (4
.mu.m) was used in all experiments, and relatively small percentage
of solids--0.07 to 0.26% (w/v) were dispersed in the organic phase,
in order to produce NCs with size
[0294] (1) Several volatile organic solvents were tested as
optional dispersed phases (ethanol, methanol, dichloromethane,
diethyl ether, acetone and acetonitrile). The best results were
obtained with acetone and acetonitrile (ACN) and therefore the
majority of encapsulation experiments were made with these
solvents.
[0295] (2) The spray drying were performed at temperatures of
60.degree. C. or 50.degree. C., when ACN was used, or at 40.degree.
C. or 30.degree. C. when the more volatile acetone was
utilized.
[0296] (3) In all the performed experiments, the yields were low
(35% at most), probably due to the volatile nature of the solvents,
leading to the appearance of "crustification" around the spraying
head (appearance of solid aggregates or crystals). Such
sedimentation blocks the vibrating membrane, and as a consequence,
leads to a poor process with low yields and high polydispersity.
This phenomenon was more pronounced when acetone was used instead
of ACN.
[0297] (4) Addition of surfactant: Tween 80 (0.06% w/v), PEG 4000
(0.03% w/v), Pluronic F-68 (0.008% w/v), or lyoprotecting materials
such as: trehalose (0.008% w/v), or sucrose (0.008% w/v), didn't
help to prevent `crustification` or diminishing the high
polydispersity of the samples. Upon their addition, the yields were
negligible, especially when Tween was used and the polydispersity
of the formed NCs was high.
[0298] (5) When low parameter spraying is applied (60% instead of
100%), the temperature of the spraying head (T.sub.h) is higher
than T.sub.in by 12.degree. C. and can lead to fusion of the formed
NCs, as displayed in FIGS. 9A-9B. When 100% spray is applied
T.sub.in is higher from T.sub.in only by 7-8.degree. C.
[0299] (6) Unlike PLGA, which produced spherical NCs in all tested
samples (FIG. 10), PEG-PLGA was found to be more sensitive to head
heating, hence in some cases, yielded amorphous NCs, as shown in
FIGS. 9A-9B. DSC measurements support this assumption, with glass
transition (Tg) values of 46.5.degree. and 31.degree. C., for PLGA
and PEG-PLGA, respectively.
[0300] (7) Reducing solids content in the sample by 2 fold (from
0.21% to 0.13%), led to improvement in yields by 2 fold (from 16%
to 35%).
[0301] (8) By sample dilution (20 ml of acetone instead of 10 ml),
we have managed to increase the amount of dispersed HSA NSs by 6
fold (from 1.6 mg to 10 mg), without reducing yields
(.about.20%).
[0302] Encapsulation of primary HSA NSs inside the organic polymers
(PEG-PLGA & PLGA) was validated using EDS (Energy Dispersive
X-ray Spectroscopy), as demonstrated in FIG. 11.
[0303] Increased amount of dispersed crosslinked HSA NSs together
with the prevention of massive crust formation around the spraying
head, were achieved by using mainly ACN as the disperse organic
phase. Furthermore, when ACN was used and the T.sub.1 was reduced
from 60.degree. C. to 50.degree. C., no crustification occurred,
and the membrane was clear throughout the entire spraying process
resulting in the formation of spherical sub-micron NCs when PLGA or
PEG-PLGA were applied (FIGS. 12A and 12B). However, still
relatively low yields (.about.30%) are obtained. This parameter
will be further examined and optimized.
Size Distribution of the Loaded NCs
[0304] Two samples with 0.1% solids content were made in ACN
(samples AO-66 and AO-68), forming PLGA NCs loaded with HSA NPs
encapsulating siRNA. We can clearly see favored formation of the
desired submicron NCs. Another sample with 1% solids (sample AO-57)
of empty 50 kDa PLGA NCs was prepared for comparison.
[0305] All PLGA NCs (empty or loaded with primary HSA NPs), were
made by Nano spray dryer B-90, operates at organic phase
(Acetonitrile, T.sub.in 50.degree. C.). The dried NCs are stored at
dark room at 4.degree. C., in sealed vials, prior to
characterization.
[0306] In order to characterized the dried NCs using Mastersizer
2000E, each sample was dispersed in water (DDW, 2 mg/ml) using
vortex, then left to stirred (with stirrer) over night in an ice
bath till a turbid homogeneous dispersion is formed.
[0307] All measurements at Mastersizer 2000E were made in a
stainless steel sample dispersion unit (120 ml volume), with an
active stirring (no need in sonication or addition of surfactant).
The results (calculated by Volume or Number), were compared to
images accepted by SEM measurements previously made for the dried
sample.
[0308] Based on size measurements by volume, made with laser
diffraction technique using Mastersizer 2000E (Malvern), it was
found that for sample AO-68, 80% of the NCs population is under 1
.mu.m (with 56% under 0.724 .mu.m) and Span value of 1.472. For
sample AO-66 better results obtained; 94% of the NCs population is
under 1 .mu.m (with 86% under 0.724 .mu.m) and Span value of 2.077.
Low span value means low polydispersity (FIGS. 13-15). For
comparison purpose, a sample with 1% solids (PLGA) content was
measured too (sample AO-57), revealing formation of bigger NCs; 94%
of the NCs population is under 2.5 .mu.m (with 42% under 1 .mu.m
and only 4% under 0.724 .mu.m). These results are encouraging and
show promise potential for the nanoencapsulation technique made
with highly diluted formulations contain--0.1% solids content.
Integrity Evaluation of Encapsulated siRNA
[0309] During the primal encapsulation process of siRNA in the HSA
NSs it has been exposed to materials with distractive potential
such as basic pH conditions and the presence of cross linker
(glutaraldehyde) who can interact with the primal amine present in
the nucleic bases: Guanine, Adenine or Cytosine. Hens, the need in
siRNA integrity evaluation arise. First, integrity evaluation of
the free siRNA (GFP-siRNA and Chol-GFP-siRNA) was made. The siRNAs
were exposed to the same conditions used for production of
crosslinked HSA NSs (3 hr at aqueous solutions with pH of 7, 8 or 9
with or without the presence of glutaraldehyde), then a fraction
from each sample was analyzed using HPLC (data not shown) and gel
retardation assay (PAGE 8%) (FIG. 16). The results, shows increase
sensitivity of siRNAs as the pH becomes more basic, while
Chol-GFP-siRNA revealed improved resistant to basic pH (8 and 9)
compared to GFP-siRNA. On the other hand, the two types of siRNA
found to be highly sensitive to the presence of the cross linker,
in all the pH tested, leading to a formation of `heavy` specie runs
slower at the gel--implying a possible distractive crosslinking
process occurred to the siRNA.
[0310] Later, siRNAs were extracted from the primal crosslinked HSA
NSs (produced in different pH conditions), and their integrity was
assessed using HPLC (data not shown) and gel retardation assay
(PAGE 8%) (FIG. 17). Furthermore, the stability of siRNAs extracted
from NCs loaded with the primal crosslinked HSA was also examined
(FIG. 15--lanes No. 9 & 10), since secondary encapsulation
process performed under heat (T.sub.1 is 50.degree. C.), using the
nano spray drying technique. Based on the PAGE results represent at
FIG. 17, we can clearly see that the siRNAs (Chol-GFP- or GFP)
encapsulated in the primal or secondary NCs, stayed intact and runs
like the untreated siRNA (lane No. 2), and no fragmentation
appeared. Based on the HPLC results, the exact amount of
encapsulated siRNA was determined. In the future, the activity of
the extracted siRNAs will need to be verified `in-vitro`.
Protocol 1--Conjugation of Ranibizumab (Lucentis) or Bevacizumab
(Avastin) to Nanospheres' Surface
MAb Wash and Quantity Determination
[0311] The Ranibizumab and bevacizumab were washed using 8.5 cm
dialysis bag (Medicell international, 12-14K) in order to remove
amino acids such as hystidine or glycine that can interact with the
LC-SMCC spacer and interfere with the MAb activation. The dialysis
bag was washed in 2 liters DDW prior to the addition of
Ranibizumab. Then about 500 .mu.l of Ab were washed in a total
volume of 3 liters PBS conc..times.10, without magnesium and
calcium diluted in DDW at a pH of 7.4. Following overnight wash,
the Ab total volume was adjusted to achieve a concentration of 2
mg/ml Ab in PBS and the Ab was centrifuged in 4.degree. C. at
14,000 rpm for 15 minutes to remove residuals of glycerol from the
dialysis bag. The Ranibizumab concentration was then determined
using a spectrophotometer at wave length of 280 nm.
MAb Activation with the Spacer LC-SMCC
[0312] The amine group (NH.sub.2) on the MAb and the ester group
(--R--COO) on the LC-SMCC were reacted to create an amide bond, the
reaction took place under rotation at 180 rpm in 4.degree. C. for 2
hours. The molar ratio of MAb to LC-SMCC was 1:100, more
specifically; 50 .mu.l of the spacer solution in DMSO (1 mg/100
.mu.l) were added to 2 mg MAb (1 ml). Final DMSO concentration did
not exceed 5%. When-needed, for Non-Activated MAb validation, 0.5
mg of residues MAb were added to about 12.5 .mu.l of DMSO to keep
the same ratio of DMSO to MAb and was incubated under the same
conditions. Following the activation Ranibizumab was centrifuged in
eppendorfs at 14000 rpm, 4.degree. C., for 10 minutes and the
supernatant was withdrawn and washed to eliminate any residues of
LC-SMCC that did not react with the MAb and to achieve a final
concentration of 1 mg/ml Ab. 2 mg (1 ml) were washed with a total
volume of 15 ml in PBSx2.5 (pH=7) using a vivaspin with 30000 MWCO
under 4000 rpm, 10-15 minutes at 4.degree. C. The process was
performed for the Non-Activated MAb as well.
NSs Preparation Process
[0313] Two formulations were manufactured according to the same
protocol. For the preparation of NPs, 75 mg Resomer 504H, 75 mg
PLGA 50-50 45000 PEG 5000 (50,000 KD), 10 mg OCA linker were
dissolved in 25 ml acetone. The organic phase was injected to 50 ml
of the aqueous phase, which contained 50 mg Solutol.RTM. RH under
stirring at a rate of 900 rpm. The stirring continued under the
same conditions for 15 minutes following injection and then the
formulation was evaporated at 37.degree. C., at a stirring rate of
30 rpm for about 1 hour.
[0314] When evaporation finished the pH was corrected to 6.8-7
using NaOH 0.1 N and the final volume was completed to 5 ml with
water for HPLC. Finally, the formulation was centrifuged for 10
minutes at 4000 rpm, room temperature to remove polymer
sediment.
NSs Incubation with Ranibizumab and Bevacizumab--Preparation of the
nanoMAb
[0315] The thiol-group on the OCA linker and the spacer melaimide
were reacted overnight at room temperature. 2 ml formulation with 2
mg MAb (at a solution concentration of 1 mg MAb/ml) were reacted in
a scintillation bottle under mild stirring (500 rpm). Non-Activated
MAb was also incubated with the NSs overnight under the same
conditions.
NanoMAbs Washing
[0316] At the next morning the samples were washed with water for
HPLC over a 300,000 MWCO vivaspin at 4000 rpm, 4.degree. C. to
eliminate residues of Mab that did not react with the NSs. The wash
volume was .times.10 from the formulation volume. The Non-Activated
NanoMAbs and NSs were washed at the same conditions. The residuals
were collected in a 15 ml polypropylene tubes and kept under
-80.degree. C. until freeze-drying, while the formulations were
concentrated to achieve the original volume of the formulations
before the wash.
NSs and nanoMAbs Characterization
[0317] 100 .mu.l from each formulation was diluted in 1000 .mu.l of
water for HPLC, filtered via 0.2 .mu.m PVDF filter and analyzed
using the Malvern Zetasizer (Malvern Instruments, Malvern UK) to
determine the zeta potential and particles diameter size.
Protocol 2--Nanoencapsulated Ranibizumab and Bevacizumab with
Crosslinked Bovine Serum Albumin (BSA)
Primary Crosslinked BSA NSs Preparation
[0318] In order to produce nanometric BSA NSs, the well known
method of pH-coacervation (also known as desolvation technique was
applied). Briefly, BSA solution, adjusted to a specific pH, was
transformed into nanoparticles by continuous (.about.1 ml/min)
addition of desolvating agent, under constant, rapid stirring
(.about.960 RPM or 40 HZ) at room temp. Addition of desolvating
agent is continued until sufficient turbidity appears (usually
between 40 to 80% (v/v) of desolvating agent), then the
crosslinking process is carried out with glutaraldehyde for at
least 2 hr at room temperature under milled shaking. After
crosslinking, desolvating agent was evaporated (37.degree. C.) and
centrifuged for 10 min at 4,000 RPM (precipitation was removed,
dried and determined gravimetrically). NSs were washed (.times.10)
with DDW, at three different cycles of centrifugation (4,000 RPM,
4.degree. C.), using vivaspin 300 kDa (Viva science).
Avastin Double Nano-Encapsulation of Non-Cross Linked BSA NSs Using
Nanospray
[0319] Bevacizumab was transferred to polysorbate 20 solution by
dialysis. (cut off 14 KDa, three time 2 hrs in 500 ml Tween-20 (0.4
mg/ml), room temperature). The Ab was then nano-encapsulsated with
BSA as described in previous report. After injection of the acetone
to the aqueous solution containing the peptide and the albumin to
elicit formation of BSA NSs during strong vortex, an aliquot of
0.75 ml of the suspension was withdrawn and incorporated
immediately to 24 ml of acetonitrile containing 16 mg PLGA (50K)
with stirring. This suspension was then evaporated using the
nanospray dryer. Final solution: DDW 1.01%, acetone 2.02%,
acetonitrile 96.97%. Solids composition: Ab 1.9%, albumin 37.7%,
PLGA 60.4%. Final solids concentration over all solution was
0.104%; Nanospray conditions: 4 .mu.m mesh, at 50.degree. C.
Encapsulation of BSA NSs Using Nanospray Dryer--Organic Mode
[0320] NCs were prepared via spray drying on the NSD B-90 operates
at `closed loop` mode, hence, N.sub.2 (g) and CO.sub.2 (g) are
flowed in the system instead of air. In all experiments gas flow
was about 120 l/min. The air soaked with volatile vapors and
humidity, transferred to a Dehumidifier unit, for drying and
condensation, then returned dry to the system in a circular path.
Spray drying was carried out at low temperatures
(Tin=30.degree.-60.degree. C.) with mesh size membrane 4 .mu.m.
Loading Efficiency of Ranibizumab and Bevacizumab onto Crosslinked
BSA NSs
[0321] For encapsulation of the Ranibizumab or bevacizumab inside
crosslinked BSA NPs, Ranibizumab or bevacizumab was added to BSA
solution prior the addition of desolvating phase. The rest of
procedure was made exactly in the same manner detailed above. After
crosslinking, the NCs were washed and the total ultrafiltrate was
collected, lyophilized and reconstituted with 500 .mu.l DDW.
Physicochemical Characterization
[0322] 100 .mu.l from each formulation was diluted in 1000 .mu.l of
water for HPLC, filtered via 0.2 .mu.m PVDF filter and analyzed
using the Malvern Zetasizer (Malvern Instruments, Malvern UK) to
determine the zeta potential and particles diameter size.
SEM and EDS
[0323] Geometry, size and surface morphology of the spray-dried NCs
(and encapsulated NSs) were observed by a High Resolution Scanning
Eelectron Microscope (HR-SEM) with High stable Schottky Field
Emission Source (Sirion, model: Quanta 200 FEI, Germany), 5 kV.
Prior to imaging, the samples were dispersed onto carbon sticky
tabs and coated with gold and palladium mixture for 90-120 sec. In
case of primary NCs dispersed in water, the samples were highly
diluted, then spattered on glass and left to evaporate overnight.
Element analysis of the specimen was made by EDS (Energy Dispersive
X-ray Spectroscopy), with X-MAX20 SDD Inca 450 EDS LN2 free
detector.
[0324] The amine groups detected by the EDS can only originate from
the presence of albumin nanocarriers inside the Nanoparticles since
no other excipient in the formula do contain amine groups
[0325] Table 8 summaries the physicochemical profile of two
formulations which were prepared by protocol-2 (Nanoencapsulated
Avastin with crosslinked bovine serum albumin (BSA); whereas Table
9 summaries the physicochemical profile of one formulation which
was prepared by protocol-1 (Conjugation of Avastin to nanospheres'
surface). Blank batch state for formulation without protein where
Ab batch state for formulation loaded with protein.
TABLE-US-00011 TABLE 8 Size and zeta potential of BSA-NPs following
Vivaspin washings Formulation Diameter of batch Ab present NSs (nm)
PDI Zeta potential (mV) Novastin-003 Blank 97.12 .+-. 1.097 0.251
45.8 .+-. 1.52 Ab 177.4 .+-. 3.161 0.083 47.9 .+-. 0.416
Novastin-006 Blank 203.4 .+-. 2.178 0.124 49.9 .+-. 0.436 Ab 212.2
.+-. 2.139 0.112 51.9 .+-. 0.513
TABLE-US-00012 TABLE 9 Size and zeta potential of INPs following
Vivaspin washings. Zeta Ab Formulation Diameter of potential
concen- % cross- batch NSs (nm) PDI (mV) tration linking Novastin-
69.62 .+-. 0.333 0.121 56.5 .+-. 6.43 005 70.09 .+-. 0.769 0.14
48.7 .+-. 0.208 8.486 95.8
[0326] Evaluation of free Ab in BSA-NP by gel electrophoresis
(protocol-2) was carried out by using the following parameters:
NuPAGE Novex Bis-Tris Mini Gels (Invitrogen), gradient 4-12%,
denaturing sample but non reduced, MOPS Running buffer, coomassie
Blue G-250 staining (limit detection 0.1 .mu.g of protein).
Electrophoresis was applied 15 days following batch formation.
[0327] As was evident from the results (results not shown), bands
density was quantified to obtain Ab concentrations. No Ab was found
in the formulations washings indicating that all the Ab have been
encapsulated in the BSA-NPs. Only low release of the encapsulated
Ab was obtained during the electrophoresis. No effective procedure
to denature the NPs and liberate the Ab has yet been found.
In-Vitro Evaluation
[0328] Uptake in A-431 cells of primary HSA NSs 2% FITC-labeled
crosslinked HSA NSs were incubated with A-431 cells (concentrations
of 1 mg/ml and 2 mg/ml per well) at 37.degree. C. over 4 h and 22
h. Based on Confocal laser scanning microscopy (CLSM), a high level
of uptake was observed after just 4 h (for the two concentrations
examined). After 22 h of incubation, no spots of NPs were detected
outside the cells (FIGS. 19A-19B).
[0329] These findings substantiate the ability of HSA NSs to
deliver loaded drug (e.g. siRNA) into target cells.
Sequence CWU 1
1
12121DNAArtificial SequenceSynthetic Construct 1ccauaaaugc
uacgaauaut t 21221DNAArtificial SequenceSynthetic Construct
2auauucguag cauuuaugga g 21321DNAArtificial SequenceSynthetic
Construct 3uaacgacgcg acgacguaat t 21421DNAArtificial
SequenceSynthetic Construct 4uuacgucguc gcgucguuat t
21524DNAArtificial SequenceSynthetic Construct 5auaucauggc
cgacaagcad tsdt 24624DNAArtificial SequenceSynthetic Construct
6ugcuugucgg ccaugauaud tsdt 24724DNAArtificial SequenceSynthetic
Construct 7auaucauggc cgacaagcad tsdt 24824DNAArtificial
SequenceSynthetic Construct 8ugcuugucgg ccaugauaud tsdt
24921DNAArtificial SequenceSynthetic Construct 9ccauaaaugc
uacgaauaut t 211021DNAArtificial SequenceSynthetic Construct
10auauucguag cauuuauggt t 211121DNAArtificial SequenceSynthetic
Construct 11ccauaaaugc uacgaauaut t 211221DNAArtificial
SequenceSynthetic Construct 12auauucguag cauuuauggt t 21
* * * * *