U.S. patent application number 15/335408 was filed with the patent office on 2018-03-08 for non-viral nanoparticle-based delivery system.
The applicant listed for this patent is THE ROYAL INSTITUTION FOR THE ADVANCEMENT OF LEARNING/MCGILL UNIVERSITY. Invention is credited to Meenakshi Malhotra, Satya Prakash.
Application Number | 20180064826 15/335408 |
Document ID | / |
Family ID | 49756122 |
Filed Date | 2018-03-08 |
United States Patent
Application |
20180064826 |
Kind Code |
A1 |
Prakash; Satya ; et
al. |
March 8, 2018 |
NON-VIRAL NANOPARTICLE-BASED DELIVERY SYSTEM
Abstract
The present invention concerns a polymeric material for the
production of a non-viral nanoparticle. The polymeric material
comprises (i) a hydrophilic linear polymer having a first end and a
second end, (iii) a cross-linkable cationic polymer covalently
bonded to the first end of the hydrophilic linear polymer, and
(iii) at least one targeting/penetrating peptide covalently
associated to the second end of the hydrophilic linear polymer.
Also disclosed herein are nanoparticles produced with these
polymeric material, processes for making the polymeric material and
the nanoparticles as well as use of the nanoparticles.
Inventors: |
Prakash; Satya; (Brossard,
CA) ; Malhotra; Meenakshi; (Montreal, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
THE ROYAL INSTITUTION FOR THE ADVANCEMENT OF LEARNING/MCGILL
UNIVERSITY |
Montreal |
|
CA |
|
|
Family ID: |
49756122 |
Appl. No.: |
15/335408 |
Filed: |
October 26, 2016 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
13916941 |
Jun 13, 2013 |
|
|
|
15335408 |
|
|
|
|
61660139 |
Jun 15, 2012 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 47/60 20170801;
A61K 47/6939 20170801; C12N 2810/851 20130101; C12N 2320/32
20130101; A61P 25/28 20180101; A61K 47/61 20170801; A61K 47/6913
20170801; A61K 48/0008 20130101; A61P 35/00 20180101; C12N
2810/6054 20130101; A61K 47/6935 20170801; A61K 48/0041 20130101;
C12N 15/111 20130101; C12N 15/88 20130101; C12N 2310/14
20130101 |
International
Class: |
A61K 48/00 20060101
A61K048/00; C12N 15/11 20060101 C12N015/11; C12N 15/88 20060101
C12N015/88 |
Claims
1 to 45. (canceled)
46. A method of treating a Polo-like kinase-1 (Plk1)-expressing
cancer in an individual in need thereof by targeted delivery of a
Plk1-targeting siRNA, said method comprising: (a) providing a
non-viral nanoparticle having and outer layer and an internal core
comprising the Plk1-targeting siRNA; and (b) administering the
non-viral nanoparticle to the individual, wherein the non-viral
nanoparticle comprises: a plurality of polymers, wherein each
polymer comprises: (i) a linear polyethylene glycol polymer (PEG)
having a first end and a second end, (ii) a cross-linkable cationic
chitosan polymer covalently associated to the first end of the
linear polyethylene glycol polymer via an ester linkage to a
monomer of the chitosan polymer, wherein the cationic chitosan
polymer has: (1) an average molecular weight of between about 10
and 200 kDa; and (2) a degree of deacetylation between about 70%
and 90%; and (iii) at least one targeting/penetrating peptide
covalently associated to the second end of the linear polyethylene
glycol polymer, wherein the at least one targeting/penetrating
peptide comprises a TAT peptide; and wherein the plurality of
cationic chitosan polymers are cross linked to each to form the
outer layer of the nanoparticle to generate the internal core; and
the Plk-targeting siRNA is in the internal core, and the non-viral
nanoparticle has an average diameter of between about 5 to 300 nm,
or between about 100 nm to 200 nm.
47. The method of claim 46, wherein the non-viral nanoparticle is
formulated for intranasal administration and the administration is
intranasal.
48. The method of claim 46, wherein the cancer is a brain cancer or
a colon cancer.
49. The method of claim 46, wherein the non-viral nanoparticle is
formulated for intravenous administration and the administration is
intravenous.
50. The method of claim 46, wherein the average molecular weight of
the cationic chitosan polymer is from about 50 to about 200
kDa.
51. The method of claim 46, wherein the degree of deactylation of
the cationic chitosan polymer is about 80%.
52. The method of claim 46, wherein the non-viral nanoparticle has
an average diameter of less than 20 nm.
53. The method of claim 46, wherein the non-viral nanoparticle is
formulated for intratumoral administration and the administration
is intratumoral.
54. A non-viral nanoparticle comprising a polymeric material
comprising: (i) a linear polyethylene glycol polymer having a first
end and a second end, (ii) a cross-linkable cationic chitosan
polymer covalently associated to the first end of the linear
polyethylene glycol polymer, and (iii) at least one
targeting/penetrating peptide covalently associated to the second
end of the linear polyethylene glycol polymer.
55. The non-viral nanoparticle of claim 54, wherein the cationic
polymer is cross-linked so as to form an internal core; and an
anionic agent entrapped in the internal core.
56. A polymeric delivery system for nucleic acids and/or anticancer
drugs, wherein the polymeric delivery system comprises
nanoparticles comprising: chitosan, polyethylene glycol and a
peptide sequence, wherein the peptide sequence comprises a cell
penetrating peptide, a protein transduction domain, and a cell
surface receptor targeting moiety, wherein the polymeric delivery
system is made by a method comprising: a. Reaction of amine groups
of chitosan with phthalic anhydride as a protection step; b.
Activation of monomethoxy polyethylene glycol (mPEG-OH) with
succinic anhydride to form mPEG-COOH; c. Activation of mPEG-COOH
with thionyl chloride to form mPEG-COCl to form an activated PEG;
d. Reaction of hydroxyl groups of chitosan to the activated
mPEG-COCl to form PEGylated chitosan; e. Reaction of monomethoxy
group of PEGylated chitosan with aluminium chloride in ethane thiol
to convert methoxy to hydroxyl group; f. Reaction of hydroxyl
terminated PEGylated chitosan with succinic anhydride to form an
activated Chitosan-PEG-COOH; g. Reaction of activated
Chitosan-PEG-COOH with amine terminated peptide (TAT/MGF/CP15)
using EDC and DMAP as a catalyst to form Chitosan-PEG-Peptide; and
h. Reaction of Chitosan-PEG-Peptide with hydrazine monohydrate to
deprotect amine groups of chitosan.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS AND DOCUMENTS
[0001] This application is a continuation of and claims priority
under 35 U.S.C. .sctn. 120 from prior co-pending U.S. application
Ser. No. 13/916,941 filed Jun. 13, 2013, which claims priority from
U.S. provisional patent application 61/660,139 filed on Jun. 15,
2012 and incorporated herein in its entirety. A sequence listing in
a computer readable format is enclosed and incorporated herein in
its entirety.
TECHNOLOGICAL FIELD
[0002] The present invention relates to the development of
nanoparticles for delivery of anionic agents such as, for example,
nucleic acids, drugs and other molecules. The non-viral
nanoparticle design involves the chemical modification of a
cationic cross-linkable polymer (such as chitosan) with a
hydrophilic linear polymer (such as polyethylene glycol) and a
targeting/penetrating peptide. The nanoparticle system can be used
for in vitro applications as well as in vivo applications.
BACKGROUND
[0003] Delivering agents specifically to a tissue, an organ or a
cell type in vivo is a complex task. However, such specific
delivery may be crucial for providing therapeutic benefits and/or
limiting undesirable side effects. In addition, some agents may
also benefit from being more efficiently translocated across a
biological membrane and/or a cellular membrane (such as, for
example, the cytoplasmic membrane or the nuclear membrane).
[0004] One application where specific drug-delivery and/or
upregulated cellular uptake is important is neurodegenerative
diseases. Neurodegenerative diseases are characterized by
progressive, age-related loss of specific subsets of neural cells,
which lead to diverse clinical phenotypes depending on the
underlying anatomical involvement. The etiology of
neurodegenerative diseases is most often multifactorial, likely a
result of gene-environmental interaction, which may lead to
diseases like Parkinson's, Alzheimer's, Huntington's, Amyotrophic
lateral sclerosis (ALS) and Spino Cerebellar Ataxia (SCA). These
diseases tend to progress slowly over the time and generally target
older population. Currently, there is no treatment available for
many of the neurodegenerative diseases that may halt the
progression of the disease. The current clinical strategy to
deliver drug/gene to the central nervous system involves surgical
interventions, which can later pose surgical complications like
fluid retention in the ventricles etc., which can have fatal
side-effects. It has been estimated that up to 98% of the newly
developed small molecules will not/cannot cross the blood-brain
barrier (BBB). Also, it is challenging to achieve sufficient
distribution and diffusion of the therapeutic drug.
[0005] Another application where specific drug-delivery and/or
upregulated cellular uptake is important is cancer. Cancer is
characterized by uncontrolled growth of a group of cells that
infests adjacent tissues and often metastasizes to other organs via
lymphatic system or blood stream. Cancer is primarily caused by
environmental factors (90-95%) and few with genetic (5-10%). The
uncontrolled growth of group of cells in the case of cancer is
usually triggered by malfunctioning of the genes that manipulate
cell's growth and differentiation. Typically the alteration in cell
growth promoting, oncogenes and cell division inhibiting, tumor
suppressive genes lead to the formation of cancer cells. The
genetic causes of cancer are usually due to gain or loss of an
entire chromosome due to errors in mitosis or changes in nucleotide
leading to mutations in the genomic DNA. Depending on the stage of
the cancer the treatment options available include surgical
removal, chemotherapy with anticancer drugs, such as
5-fluorouracil, oxaliplatin and leucovorin, radiation therapy,
immunotherapy and hormone block therapy with drugs like cetuximab
and panitumumab. However, it has been shown that cancers with
genetic origin are not benefited with these chemotherapies.
Moreover, the toxicity and side effects have severely limited the
safety and effectiveness of these methods.
[0006] RNA interference has been demonstrated as an effective/novel
therapeutic modality in vivo for the reduction of pathological
molecules in neurons, leading to significant efficacy in animal
models of Alzheimer's disease, ALS, anxiety, depression,
encephalitis, Huntington's disease, neuropathic pain and
spinocerebellar ataxia. In addition, siRNAs targeted against
proliferation-associated signal transduction pathways, which can
halt the tumor progression in animal models, is also emerging as an
appealing approach for cancer therapy. SiRNA mediated gene therapy
is further being explored by using it in combination with
antineoplastic agents as a combinatorial therapy towards cancer
treatment.
[0007] The therapeutic delivery across BBB and physiological
barriers of small molecules like siRNA/nucleic acids/proteins and
drugs is challenging or rather ineffective owing to their
instability in physiological conditions, improper cellular
distribution, low bioactivity, high dosage requirement, and the
necessity for continuous long-term infusions. Moreover, off-target
effects of siRNA have shown to induce and activate immune responses
through Toll-like receptor (TLR)-dependent and TLR independent
mechanisms, which can cause toxicological effects due to the
activation of interferons (IFNs) and inflammatory cytokines.
Though, this strategy can be used as a therapeutic option for
cancer therapy, it can also lead to unwanted toxicological effects
on non-cancerous cells. The application of a novel therapeutic
modality highly depends on the development of an efficient and
clinically feasible means of safe administration. Recent
advancements in siRNA mediated gene therapy is focusing on the
incorporation of specific nucleotide chemistries in the siRNA
sequence, such as 2'-O-methyl-modified nucleotides, in order to
improve its pharmacologic and nuclease resistant properties and low
immuno-stimulatory effect. However, to streamline the systemic
delivery of siRNAs in vivo to a specific target tissue, a
site-directed delivery strategy is essential.
[0008] Various commercially available delivery/transfection
reagents do fulfill the need of siRNA delivery in substantially
lower doses than compared to siRNA delivered alone, but they lack
to emphasize on issues such as target specificity, cytotoxicity,
immunological responses, stable systemic delivery and off-target
effects of these reagents. Moreover, the efficacy of most of these
commercially available transfection reagents is limited to in vitro
use. In addition to transfection reagents, viral gene therapy
vectors have shown to be most effective but concerns regarding
their safety, immunogenicity has limited their use. Moreover, for
in vivo applications, delivery via systemic route targets multiple
sites, which may not be an ideal deal for many biomedical
applications. Thus, development of a delivery vehicle that can
address these issues and identify cell-specific receptors, with an
ability to distinguish between diseased and normal cells and
efficiently delivers a sufficient dose of siRNA to the
intracellular compartment of the cell type of interest within the
target tissue will be an effective approach to overcome the
limitations of currently used therapeutics in in vivo models.
[0009] Cationic biodegradable polymers, such as polyethyleneimine
(PEI), are commonly used for siRNA transfection, providing a
positively charged vehicle to carry the negatively charged siRNA
into the target cells. Most of the nanoparticles that are widely
being accepted as an alternative approach to gene delivery are
developed from cyclodextrin polycations, poly-L Lysine,
polyamidoamines, chitosan, quantum dots and liposomes to deliver
siRNA for on-target gene silencing. However, transfection
frequency, cytotoxicity, serum stability, and active targeting are
some of the major concerns associated with non-viral mode of
delivery. To overcome the aforementioned effects of nanoparticles,
they are surface coated with PEG to eliminate phagocyte capture and
nonspecific immune stimulation, which provides better stability and
extended blood circulation with low toxicity. Conjugating siRNA
with cholesterol and transfection enhancers, such as penetratin1,
provides substantial advantage for in vivo delivery and bioactivity
of siRNA.
[0010] Lipid nanoparticles have been used for drug delivery
targeting brain. However, such formulations lack active targeting
to the tissue of interest. It has been proposed to use monoclonal
antibody as a targeting moiety on a non-viral liposome
nanoparticles targeting brain. However, the high molecular weight
of antibodies makes the overall size of the nanoparticles big in
size, which can therefore be a hindrance to achieve optimal
biodistribution at the targeted site. A recent addition to the art
is the use of PEI and nucleic acid complex for intraventricular
stereotactic screening of stem cells of the brain as a treatment
for neurodegenerative diseases and demyelinating diseases. However,
PEI as has been known to be a non-biodegradable and toxic could be
replaced by biodegradable nanocomplexes with cell-targeting
ligands.
[0011] In summary, it would be highly desirable to be provided with
a delivery system for specifically delivering agents to a tissue
(or a group of tissues), an organ (or a group of organs) or a cell
type (or a group of cell types). The cellular specificity of the
delivery system could be used to lower the dose of the agent as
well as limit, if any, the undesirable effects associated with the
agent. The delivery system is preferably not based on a viral
system and is not capable of reproducing once it enters a cell. It
would also be desirable that the delivery system be used in vitro
for research purposes, ex vivo for cellular therapy applications as
well as in vivo for therapy or diagnostics. In some embodiments, it
would be desirable to be provided with a delivery system that is
also capable of more efficiently translocating across a biological
membrane and/or a cellular membrane (such as, for example, the
cytoplasmic membrane or the nuclear membrane).
SUMMARY
[0012] According to a first aspect, the present invention provides
a polymeric material of a non-viral nanoparticle. The polymeric
material comprises (i) a linear polyethylene glycol polymer having
a first end and a second end, (ii) a cross-linkable cationic
chitosan polymer covalently associated to the first end of the
linear polyethylene glycol polymer, and (iii) at least one
targeting/penetrating peptide covalently associated to the second
end of the linear polyethylene glycol polymer. In one embodiment,
the linear polyethylene glycol polymer has an average molecular
weight between about 1000 to about 5 000 Da. In another embodiment,
the linear polyethylene glycol polymer has an average molecular
weight of about 2 000 Da. In still another embodiment, the
cross-linkable cationic chitosan polymer has an average molecular
weight of about 10 to about 200 000 Da. In still a further
embodiment, the average molecular weight of the cross-linkable
cationic chitosan polymer is about 1 00 000 Da. In one embodiment,
the cross-linkable cationic chitosan polymer has a degree of
deacetylation of about 75 to about 85%. In another embodiment, the
degree of deacetylation of the cross-linkable cationic chitosan
polymer is about 80%. In yet another embodiment, the at least one
targeting/penetrating peptide can bind to a cell surface or
cell-associated receptor, such as, for example, a targeting peptide
having an amino acid sequence comprising
NH.sub.2-YQPPSTNKNTKSQRRKGSTFEEHK-NH.sub.2 (MGF) (SEQ ID NO: 1), a
targeting peptide having an amino acid sequence comprising
NH.sub.2-VHLGYAT-NH.sub.2 (CP15) (SEQ ID NO: 2) and/or a targeting
peptide having an amino acid sequence comprising
NH.sub.2-VPWMEPAYQRFL-NH.sub.2 (P160) (SEQ ID NO: 3). In another
embodiment, the at least one targeting/penetrating peptide can
facilitate the translocation of the non-viral particle across a
biological or a cellular membrane, such as, for example, a
penetrating peptide having an amino acid sequence comprising
NH.sub.2-RKKRRQRRR-NH.sub.2 (TAT) (SEQ ID NO: 4).
[0013] In a second aspect, the present invention provides a process
for making the polymeric material described herein. Broadly, the
process comprises (i) reacting the first end of a linear
polyethylene glycol polymer derivative with the cross-linkable
cationic chitosan polymer to form a covalent bond between the
cross-linkable cationic chitosan polymer and the linear
polyethylene glycol polymer, and (ii) reacting the second end of
the linear polyethylene glycol polymer derivative with the at least
one targeting/penetrating peptide to form a covalent bond between
the at least one targeting/penetrating peptide and the linear
polyethylene glycol polymer. In an embodiment, the process further
comprises, in step (i) prior to reacting the first end, protecting
at least one amine group of the cross-linkable cationic chitosan
polymer. In still another embodiment, the process further
comprises, after step (ii), deprotecting the at least one amine
group of the cross-linkable cationic chitosan polymer. In an
embodiment, the linear polyethylene glycol polymer derivative is a
linear monomethyl ether polyethylene glycol (mPEG-OH). In another
embodiment, the process further comprises, in step (i) prior to
reacting the first end, activating the first end of the linear
polyethylene glycol monomethyl ether by reacting the polyethylene
glycol monomethyl ether with succinic anhydride to form mPEG-COOH
and reacting the mPEG-COOH with thionyl chloride to form a
mPEG-COCl. In still another embodiment, the process further
comprises, in step (i), reacting the mPEG-COCl with the
cross-linkable cationic chitosan polymer to form a PEGylated
chitosan. In yet another embodiment, the process further comprises,
in step (ii) prior to reacting the second end, activating the
second end of the polyethylene glycol monomethyl ether by reacting
the methyl ether group of the polyethylene glycol with aluminium
chloride in ethanethiol to convert the methyl ether group to a
hydroxyl group and reacting the hydroxyl group of the polyethylene
glycol with succinic anhydride to form an activated PEG-COOH. In an
embodiment, the process further comprises, in step (ii), reacting
the activated PEG-COOH with the at least one targeting/penetrating
peptide to form the polymeric material.
[0014] According to a third aspect, the present invention provides
a non-viral nanoparticle. The non-viral nanoparticles comprises (i)
a plurality of polymeric materials as described herein or produced
by the process described herein, wherein the cationic polymer is
cross-linked so as to form an internal core; and (ii) an anionic
agent entrapped in the internal core. In an embodiment, the anionic
agent is a nucleic acid molecule, such as, for example, a short
interfering RNA (siRNA). In an embodiment, the non-viral
nanoparticles have a diameter of less than 5 nm. In another
embodiment, the non-viral nanoparticles have a diameter between 5
and 10 nm. In a further embodiment, the non-viral nanoparticles
have a diameter between 100 and 200 nm. In still another
embodiment, the non-viral nanoparticle have a diameter between
about 5 nm to about 100 nm or between about 50 nm to about 300 nm.
In yet another embodiment, the plurality of polymeric materials
comprises a first polymeric material having a first
targeting/penetrating peptide that can bind to a cell surface or
cell-associated receptor and a second polymeric material having a
second targeting/penetrating peptide that can facilitate the
translocation of the non-viral particle across a cellular membrane.
In such embodiment, the first targeting/penetrating peptide can
have an amino acid sequence comprising
NH.sub.2-YQPPSTNKNTKSQRRKGSTFEEHK-NH.sub.2 (MGF) (SEQ ID NO: 2)
and/or the second targeting/penetrating peptide can have an amino
acid sequence comprising NH.sub.2-RKKRRQRRR-NH.sub.2 (TAT) (SEQ ID
NO: 4).
[0015] According to a fourth aspect, the present invention provides
a process for making a non-viral nanoparticle. Broadly, the process
comprises (i) admixing an anionic agent with a cross-linker to form
a first solution; and (ii) adding the first solution to a second
solution comprising a plurality of polymeric materials described
herein or produced by the process described herein so as to form
the non-viral nanoparticle. In an embodiment, the cross-linker is
sodium tripolyphosphate (TPP). In another embodiment, the anionic
agent is a nucleic acid molecule, such as, for example, a short
interfering (RNA). In an embodiment, the non-viral nanoparticle has
a diameter between about 5 nm to about 100 nm or between about 50
nm to about 300 nm. In an embodiment, the non-viral nanoparticles
have a diameter of less than 5 nm. In another embodiment, the
non-viral nanoparticles have a diameter between 5 and 10 nm. In a
further embodiment, the non-viral nanoparticles have a diameter
between 100 and 200 nm. In still another embodiment, the plurality
of polymeric materials comprises a first polymeric material having
a first targeting/penetrating peptide that can bind to a cell
surface or cell-associated receptor and a second polymeric material
having a second targeting/penetrating peptide that can facilitate
the translocation of the non-viral particle across a cellular
membrane. In such embodiment, the first targeting/penetrating
peptide can have an amino acid sequence comprising
NH.sub.2-YQPPSTNKNTKSQRRKGSTFEEHK-NH.sub.2 (MGF) (SEQ ID NO: 1)
and/or the second targeting/penetrating peptide can have an amino
acid sequence comprising NH.sub.2-RKKRRQRRR-NH.sub.2 (TAT) (SEQ ID
NO: 4).
[0016] According to a fifth aspect, the present invention provides
a method of delivering an anionic agent to a cell. Broadly the
method comprises contacting the non-viral nanoparticle described
herein or produced by the process described herein with the cell
under conditions sufficient for allowing the anionic agent to enter
the cell. In an embodiment, the cell is in vitro. In another
embodiment, the cell is to be introduced into an individual in need
thereof. In still another embodiment, wherein the cell is in an
individual in need thereof. In an embodiment, the non-viral
nanoparticle is formulated for intranasal administration prior to
being administered to the individual, such as, for example, as an
intranasal drop. In another embodiment, the method is for the
delivery of the agent to the brain. In another embodiment, the
method is for the prevention, treatment and/or alleviation of
symptoms associated to a neurodegenerative disease or a brain
cancer. In some embodiments, the neurodegenerative disease is
selected from the group consisting or spinocerebellar ataxia,
Huntington's disease, Parkinson's disease, Alzheimer's disease,
dementia and amyotrophic lateral sclerosis. In other embodiment,
the non-viral nanoparticle is formulated for intravenous
administration prior to being administered to the individual. In
another embodiment, the method is for the prevention, treatment
and/or alleviations of symptoms associated with a proliferative
disease, such as, for example cancer (e.g. colon cancer, breast
cancer or brain cancer).
[0017] Throughout this application, various terms are used and some
of them are more precisely defined herein.
[0018] Anionic agent. The anionic agent bears a net negative charge
(at a physiological pH) and can be formulated inside the internal
core formed by the cross-linked cationic polymer. In one
embodiment, it is a therapeutic agent which can mediate a
therapeutic/biological action on a cell or in an organism. In
another embodiment, it is a diagnostic agent which can specifically
be located in a cell or in a tissue for diagnostic purposes (e.g.
imaging for example). The anionic agent can be a nucleic acid
molecule, such as a RNA or a DNA. In some embodiments, the anionic
agent can be, for example an small interfering RNA (siRNA), short
hairpin (shRNA), ribozymes, micro RNA (miRNA), triplex
oligonucleotides, antisense oligonucleotides, plasmid DNA (pDNA) as
well as combinations thereof. In another embodiment, the anionic
agent is a small chemical entity, such as a drug (e.g. an
anti-cancer drug).
[0019] Cross-linkable cationic polymer. This polymer bears a net
positive and is capable of being cross-linked. Its
cross-linkability may be inherent to the polymer (e.g. the polymer
may itself contain cross-linkable groups). However, the polymer can
also be modified to augment its cross-linkability. The
cross-linkable moieties of the polymer are preferably amino groups.
This polymer is preferably biocompatible. This polymer, in some
embodiments, can be considered hydrophobic or slightly soluble in
water. Once integrated in the polymeric material or the
nanoparticle, the polymer preferably is not cytotoxic. In an
embodiment, the cationic polymer is chitosan.
[0020] Chitosan. Chitosan is a polysaccharide obtained by
N-deacetylation of chitin. In industrial scale procedures, chitosan
is obtained from chitin by alkali treatment of crustacean shells.
Chitosan is also present in nature in the cell walls of some fungi
and algae and in insects. Chitosan is mainly composed of
.beta.-1,4-linked D-glucosamine units with a variable content of
N-acetyl-D-glucosamine units. The percentage of
N-acetyl-D-glucosamine units is defined as the degree of
N-acetylation of chitosan ("DA"), while the percentage of
D-glucosamine units is also called the degree of deacetylation
("DDA") of chitosan. Most commercial preparations of chitosan are
characterized by dda values between 70 and 99%. Native chitosan
molecules, as isolated from natural organisms or obtained after
alkaline N-deacetylation of chitin, are of high molecular weight
(in the range of millions of daltons) with degrees of
polymerization reaching several thousand units. While various
applications were described for such high molecular weight
chitosans (HMWC), for most applications a narrower, optimal range
of molecular weight is under consideration. Chitosan polymers with
shorter than native chains are often divided into low molecular
weight chitosans (LMWC), with a range of molecular weight roughly
between 5 kDa and 100 kDa and chitooligosaccharides (or chitosan
oligosaccharides; CHOS) with a lower limit of 0.4 kDa (glucosamine
dimer) while the higher limit is less defined (5 to 10 kDa). The
CHOS are fully soluble in water and are essentially prepared as
undefined mixtures of oligomeric molecules of various molecular
weights and degrees of N-acetylation. Chitosan is a cationic
polymer due to the presence of amine groups at the C6 position of
its pyranose ring and, in its native unmodified state, is only
soluble in mild acidic conditions.
[0021] Crosslinker. The crosslinker is a compound that promotes or
regulates the cross-linking between different groups of the
cationic polymer chains, linking them together to create an
internal core. In an embodiment, the crosslinker is sodium
tripolyphosphate (TPP).
[0022] Hydrophilic linear polymer. This polymer is linear in nature
(it lacks any branching and only possesses two opposite terminal
ends). The hydrophilic linear polymer has at least two distinct and
different end, each covalently associated with either the cationic
polymer or the targeting/penetrating peptide. It also acts as a
linker to camouflage the cross-linked cationic polymer internal
core. It also serves to present the targeting/penetrating peptide
to the cell. This polymer is preferably hydrophilic to facilitate
cellular membrane translocation of the non-viral nanoparticle. This
polymer can be biophobic (does not allow on its own, the
association to a cell). This polymer is preferably biologically
inert.
[0023] Internal core. This section of the nanoparticle is formed
once the cationic polymer has been cross-linked. The internal core
is camouflaged under a layer of hydrophilic linear polymer and
contains the anionic agent.
[0024] Non-viral nanoparticles. As used in the context of this
invention, the term "non-viral nanoparticles" refer to particles in
the nanometer range. These particles are considered to be
"non-viral" because, even though in some embodiments they are can
contain nucleic acid molecules, the particles are not capable of
replicating once they have entered a host cell. These nanoparticles
are generally of a spherical shape. In some embodiments, the
average diameter of the non-viral nanoparticle can be between about
5 to about 100 nm or to about 200 nm. In other embodiments, the
average diameter of the non-viral diameter can between about 50 to
about 300 nm. In an embodiment, the non-viral nanoparticles have a
diameter of less than 5 nm. In another embodiment, the non-viral
nanoparticles have a diameter between 5 and 10 nm. In a further
embodiment, the non-viral nanoparticles have a diameter between 100
and 200 nm.
[0025] Pharmaceutical composition. As used herein, "pharmaceutical
composition" means therapeutically effective amounts (dose) of the
non-viral nanoparticles together with pharmaceutically acceptable
diluents, preservatives, solubilizers, emulsifiers, adjuvants
and/or carriers. Such compositions are liquids or lyophilized or
otherwise dried formulations and include diluents of various buffer
content (e.g., Tris-HCl, acetate, phosphate), pH and ionic
strength, additives such as albumin or gelatin to prevent
absorption to surfaces, and detergents (e.g. Tween 20.TM., Tween
80.TM., Pluronic F68.TM., bile acid salts). The pharmaceutical
composition can comprise pharmaceutically acceptable solubilizing
agents (e.g. glycerol, polyethylene glycol), anti-oxidants (e.g.
ascorbic acid, sodium metabisulfite), preservatives (e.g.
thimerosal, benzyl alcohol, parabens), bulking substances or
tonicity modifiers (e.g. lactose, mannitol), covalent attachment of
polymers such as polyethylene glycol to the protein, complexation
with metal ions, or incorporation of the material into or onto
particulate preparations of polymeric compounds such as polylactic
acid, polyglycolic acid, hydrogels, etc., or onto liposomes,
microemulsions, micelles, unilamellar or multilamellar vesicles,
erythrocyte ghosts, or spheroplasts. Such compositions will
influence the physical state, solubility, stability, rate of in
vivo release, and rate of in vivo clearance.
[0026] Polyethylene Glycol. Polyethylene glycol has the formula
H(OCH.sub.2CH.sub.2).sub.nOH, wherein n is greater than or equal to
4, with a molecular weight of up to about 20000 Daltons. Various
derivatives of polyethylene glycol may substitute for the H or OH
end groups, forming, for example, polyethylene glycol ethers (e.g.
PEG-O--R, PEG-O--CH.sub.3; CH.sub.3-PEG-OH), 2,4-dinitrophenyl
ethers of PEG), polyethylene glycol esters (e.g.
PEG-O.sub.2C(CH.sub.2).sub.14CH.sub.3;
PEG-O.sub.2CCH.sub.2CH.sub.2CO.sub.2-atropine), polyethylene glycol
amides (e.g. PEG-O.sub.2C(CH.sub.2).sub.7CONHR;
mPEG-O.sub.2CCH.sub.2CH.sub.2CONH(CH.sub.3)CHCH.sub.2C.sub.6H.sub.5;
PEG-O.sub.2CCH.sub.2CH.sub.2CONHCH.sub.2CH.sub.2-NAD+),
polyethylene glycol amines (e.g. PEG-NH.sub.2;
PEG-NH(CH.sub.2).sub.6NH.sub.2; PEG-OCH.sub.2CH.sub.2NH.sub.2;
mPEG-NH.sub.2), polyethylene glycol acids (e.g.
PEG-O.sub.2C(CH.sub.2).sub.2CO.sub.2H; PEG-O--CH.sub.2CO.sub.2H;
PEG-O.sub.2C--(CH.sub.2).sub.7--CO.sub.2H), polyethylene glycol
aldehydes (e.g. PEG-O-CH.sub.2--CHO), and electrophilic derivatives
(e.g. PEG-Br; PEG-OSO.sub.2CH.sub.3; PEG-O). Various phenyl
moieties can also be substituted for the H or OH of PEG, such as
the 2,4-dinitrophenyl ether of PEG mentioned above. The particular
polyethylene glycol derivatives listed above are exemplary only,
and the invention is not intended to be limited to those particular
examples.
[0027] Prevention, treatment and alleviation of symptoms. These
expressions refer to the ability of a method or an agent to limit
the development, progression and/or symptomology of a specific
disorder or pathology. The prevention, treatment and/or
alleviations of symptoms associated with a neurodegenerative
disease can encompass the limitation of neurodegeneration (e.g. by
reducing the apoptosis of neuronal cells). The prevention,
treatment and/or alleviation of symptoms of proliferative disease
can encompass the reduction of proliferation of the cells (e.g. by
reducing the total number of cells in an hyperproliferative state
and/or by reducing the pace of proliferation of cells). Symptoms
associated with proliferation-associated disorder include, but are
not limited to: local symptoms which are associated with the site
of the primary cancer (such as lumps or swelling (tumor),
hemorrhage, ulceration and pain), metastatic symptoms which are
associated to the spread of cancer to other locations in the body
(such as enlarged lymph nodes, hepatomegaly, splenomegaly, pain,
fracture of affected bones, and neurological symptoms), and
systemic symptoms (such as weight loss, fatigue, excessive
sweating, anemia and paraneoplastic phenomena).
[0028] Proliferation-associated disorders. These disorders form a
class of diseases where cells proliferate more rapidly, and usually
not in an ordered fashion. The proliferation of cells cause a
hyper-proliferative state that may lead to biological dysfunctions,
such as the formation of tumors (malignant or benign). One of the
proliferation-associated disorder is cancer. Also known medically
as a malignant neoplasm, cancer is a term for a large group of
different diseases, all involving unregulated cell growth. In
cancer, cells divide and grow uncontrollably, forming malignant
tumors, and invade nearby parts of the body. The cancer may also
spread to more distant parts of the body through the lymphatic
system or bloodstream. In an embodiment, the cancer is a carcinoma
(such as a colon carcinoma or a breast carcinoma). In another
embodiment, the cancer is a glioma (such as a brain glioma). In
another embodiment, the cancer is a colon cancer, a breast cancer
and/or a brain cancer.
[0029] Targeting/penetrating peptide. This peptide is covalently
associated with the hydrophilic linear polymer and serves to
provide specificity for the delivery of the anionic agent. In some
embodiments, the targeting/penetrating peptide is hydrophilic in
nature. For example, a targeting/penetrating peptide can have both
polar and apolar ends or it can have an alternating pattern of
hydrophilic and hydrophobic amino acids. The targeting/penetrating
peptide is preferably at least 3 amino acid-long and less than 40
amino-acids long. In one embodiment, the targeting/penetrating
peptide preferably and specifically binds to a cellular structure
(such as a cellular receptor having at least one portion either
embedded in the cellular membrane or protruding from the cellular
membrane). When the anionic agent is to be administered to a
specific type of cells, the targeting/penetrating peptide
specifically binds to this specific type of cells. Alternatively or
complementarily, the peptide may also possess a biological
characteristic that enables it to penetrate a certain cellular
structure. Specific targeting/penetrating peptides are known in the
art and can be, for example, capable of penetrating a cellular
membrane (such as the cytoplasmic membrane or the nuclear
membrane). Some targeting/penetrating peptide can be specific or
derived from a protein transduction domain. Other
targeting/penetrating peptide can be specific or derived from a
growth factor or a hormone. An exemplary targeting/penetrating
peptide can be a blood-brain-barrier (BBB)-permeant,
amyloid-targeting/penetrating peptide such as KKLVFFA.xi.KGC (as
presented in U.S. Pat. No. 7,803,351). Other exemplary
targeting/penetrating peptides include, but are not limited to TAT,
MGF, CP15 and P160 as well as combinations derived therefrom (for
example a TAT/MGF combination).
[0030] Therapeutically effective amount. A "therapeutically
effective amount" as used herein refers to that amount which
provides a therapeutic effect for a given condition and
administration regimen.
BRIEF DESCRIPTION OF THE DRAWINGS
[0031] Having thus generally described the nature of the invention,
reference will now be made to the accompanying drawings, showing by
way of illustration, preferred embodiments thereof.
[0032] FIG. 1 shows a possible synthetic scheme for preparing
peptide-tagged PEGylated chitosan polymer.
[0033] FIG. 2 shows FTIR spectra of commercially available chitosan
(CS) (plot a) and 2-N-phthaloylated chitosan: v.sub.max/cm.sup.-1
3200-3400 (OH), 1774, 1710 (carbonyl anhydride), 1150-1000
(pyranose), and 720 (arom) (plot b).
[0034] FIG. 3 shows FTIR spectra of polyethylene glycol monomethyl
ether (mPEG-OH) (plot a) and carboxylated mPEG (mPEG-COOH):
v.sub.max/cm.sup.-1 1732 (C.dbd.O), 2878 (C--H stretching), 1100
(C--O stretching) (plot b).
[0035] FIG. 4 shows FTIR spectra of 2-N-phthaloyl chitosan-O-PEG:
v.sub.max/cm.sup.-1 2873 (C--H stretching), 1068 (C--O stretching)
of PEG, 1774, 1710 (carbonyl anhydride) and 720 (arom) of
phthalimido group on chitosan (plot a); TAT tagged PEGylated
phthaloyl chitosan (2-N-phthaloyl chitosan-O-PEG-CONH-TAT):
v.sub.max/cm.sup.-1 2919 (C--H stretching), 1067 (C--O stretching)
of PEG, 1774, 1710 (carbonyl anhydride) and 720 (arom) of
phthalimido group on chitosan, 1659 (amides) in TAT peptide (plot
b); deprotected TAT tagged PEGylated chitosan
(chitosan-O-PEG-CONH-TAT): v.sub.max/cm.sup.-1 2918 (C--H
stretching), 1061 (C--O stretching) of PEG, 1644 amides of TAT
peptide, and 1543 (amides) in chitosan (plot c).
[0036] FIG. 5 shows .sup.1H NMR spectra of TAT peptide (plot a);
deprotected TAT tagged PEGylated chitosan (CS-O-PEG-CONH-TAT) (plot
b); TAT tagged PEGylated phthaloyl chitosan (PHCS-O-PEG-CONH-TAT)
(plot c); PEGylated phthaloyl chitosan (PHCS-O-PEG) (plot d); and
phthaloylated chitosan (PHCS) (plot e).
[0037] FIGS. 6A-6F show transmission electron microscopy (TEM)
image of PEGylated chitosan polymer, magnification: 53 800.times.,
scale bar: 500 nm (FIG. 6A); TAT/MGF tagged PEGylated chitosan
polymer, magnification: 53 800.times., scale bar: 500 nm (FIG. 6B);
acid treated (pH 5.5) TAT/MGF tagged PEGylated chitosan polymer,
magnification: 70 700.times., scale bar: 500 nm (FIG. 6C); empty
TAT/MGF tagged PEGylated chitosan nanoparticles, magnification: 162
000.times., scale bar: 100 nm (FIG. 6D); unmodified chitosan-siRNA
nanoparticles, magnification: 122 000.times., scale bar: 100 nm
(FIG. 6E); and chitosan-PEG-TAT/MGF-siRNA nanoparticles,
magnification: 302 000 .times., scale bar: 100 nm (FIG. 6F).
[0038] FIG. 7 shows mouse neuroblastoma (Neuro2a) cells transfected
with modified chitosan-PEG TAT-siGLO nanoparticles. siGLO (red) is
a scrambled siRNA, which is tagged with a Cy3 dye, which upon
transfection indicates the delivery of siRNA.
[0039] FIG. 8 shows cytotoxicity study on mouse neuroblastoma cells
(Neuro2a) with various treatment using the MTS assay. The results
indicate that nanoparticles with chitosan-PEG-TAT and chitosan-PEG
formulation showed minimal toxicity to cells as compared to
unmodified chitosan nanoparticles. The absorbance was measured at
490 nm using a multiplate cell counter. Control:, Chitosan:, TPP:,
siGLO: scrambled siRNA alone, Chitosan-TPP-siGLO:,
Chitosan-PEG-TPP-siGLO:, Chitosan-PEG-TAT-TPP-siGLO:.
[0040] FIG. 9 shows western blot analysis of ataxin protein
performed 24 and 48 hours post-transfection. Samples A and C are
positive controls with nanoparticles containing no siRNA and
scrambled siRNA (siGLO) respectively. Sample B contains
nanoparticles with Ataxin1-siRNA. Silencing is observed after 48
hours for sample B. Actin is used as a protein loading control. The
lower panel in this figure provides control results for actin.
[0041] FIGS. 10A-10B represent histopathological images of brain
tissues (cerebral cortex (first row) and cerebellum (second row))
four hours after receiving the nanoparticle formulation of
biotin-siRNA at various doses: 0.25 mg/kg, 0.5 mg/kg, 1 mg/kg, 2
mg/kg and PBS only (control) (FIG. 10A). The circled regions in
column (B), identified with DAB staining, indicate the presence of
biotin-tagged scrambled siRNA in neurons observed. Magnification
400.times.. FIG. 10B graphically illustrates Quantitative analysis
of the stained areas of FIG. 10A in tissues using the Image J
software. Results are shown for the cerebral cortex (dark gray) and
the cerebellum (light gray) as percentage of area positive for DAB
staining in function of siRNA dose. The graph shows a
representative result of independent readings from two animals in
each group (n=2) mean.+-.s.d. ***P<0.01 was considered highly
significant based on Tukey's post-hoc analysis, when compared with
other groups.
[0042] FIGS. 11A-11B represent histopathological images (cerebral
cortex (first row) and cerebellum (second row)) with nanoparticles
carrying 0.5 mg/kg of biotin-siRNA at different time points: 4 hrs,
16 hrs, 24 hrs and 48 hrs after the administration of the
biotin-siRNA (FIG. 11A). The circled regions in column A,
identified with DAB staining, indicate the presence of
biotin-tagged scrambled siRNA in neurons delivered by
nanoparticles. Magnification 400.times.. FIG. 11B graphically
illustrates Quantitative analysis of the stained areas in tissues
of FIG. 11A using the Image J software. Results are shown for the
cerebral cortex (dark grey) and the cerebellum (light gray) as
percentage of area positive for DAB staining in function of time
elapsed since the administration of the biotin-siRNA. The graph
shows a representative result of independent readings from two
animals in each group (n=2) mean.+-.s.d. ***P<0.01 was
considered highly significant based on Tukey's post-hoc analysis,
when compared with other groups.
[0043] FIGS. 12A-12B represent biodistribution (through
histopathological staining) of various organs collected 4 hrs
following administration of the nanoparticle formulation containing
biotin-siRNA dose at 0.5 mg/kg in animals. In FIG. 12A, results are
shown for treated animals (first two columns) and control animals
(treated with saline, last two columns) in the cerebral cortex, the
cerebellum, the lungs, the heart, the kidney the liver and the
stomach. The results show a maximum delivery of biotin-siRNA in the
brain (cerebral cortex and cerebellum), with a lesser extent in the
heart, kidney, liver lungs and stomach. Magnification 400.times..
FIG. 12B shows quantitative analysis of the stained areas of FIG.
12A in tissues using the Image J software. Results are shown as
percentage of area positive for DAB staining in function of organ
(cerebral cortex, the cerebellum, the lungs, the heart, the kidney
the liver and the stomach) at various doses (from left to right for
each organ 0.25 mg/kg, 0.5 mg/kg, 1 mg/kg and 2 mg/kg), compared to
the untreated control receiving 0.85% w/v NaCl. The graph shows a
representative result of independent readings from two animals in
each group (n=2) mean.+-.s.d. ***P<0.01 was considered highly
significant based on Tukey's post-hoc analysis, when compared with
other groups.
[0044] FIGS. 13A-13B represent enlarged DAB stained
histopathological sections of cerebral cortex (FIG. 13A) and
cerebellum (FIG. 13B) with nanoparticle formulation complexing
biotin tagged scrambled siRNA at dose 0.5 mg/kg. The cerebral
neurons in FIG. 13A and Purkinje cells in cerebellum in FIG. 13B
show a clear brown staining indicating the presence and successful
delivery of biotin tagged scrambled siRNA in these cells by
nanoparticles administered via intranasal route. The brown color
obtained by DAB staining in FIGS. 13A and 13B represents presence
of biotin-tagged scrambled siRNA in neuronal cells of cerebral
cortex and Purkinje cells of cerebellum respectively, as delivered
by nanoparticles. Magnification 400.times..
[0045] FIGS. 14A-14F represent histopathological images of various
organ tissues collected 4 hrs following intranasal administration
of multifunctional siRNA/nanoparticle formulation containing
biotin-siRNA at a dose of 0.5 mg/kg. The tissues were stained with
the TUNEL apoptosis assay. Results are shown for the cerebral
cortex (FIG. 14A), cerebellum (FIG. 14B), heart (FIG. 14C), lungs
(FIG. 14D), kidneys (FIG. 14E), liver (FIG. 14F) and stomach (FIG.
14G). As indicated in these images, the absence of TUNEL-specific
staining represents no apparent cell toxicity/apoptosis in any of
the tissues. Magnification 400.times..
[0046] FIG. 15 shows a schematic diagram of a proposed mechanism of
cellular uptake of a nanoparticle device comprising peptide tagged
PEGylated chitosan nanoparticles carrying either nucleic acid
(siRNA/DNA/RNA) or an anticancer drug as a therapeutic
molecule.
[0047] FIG. 16 shows the results associated with the percentage
PSN1 gene knockdown upon administration of the nanoparticle
composition. Results are shown as relative expression (in function
of GADPH) of PSN1 for animals treated with the nanoparticles
bearing a PSN1 siRNA (left column) and for control (untreated)
animals (right column). The results indicate a 21.34% reduction in
PSN1 gene expression with nanoparticles carrying siRNA against PSN1
gene, when compared with untreated control. The graph shows a
representative result (average of n=4.+-.S.E.).
[0048] FIGS. 17A-17E show the result of serum analysis performed on
animals treated with the nanoparticles bearing the PSN1 siRNA or a
control. Results are shown for ALP levels (U/L) (FIG. 17A), AST/ALT
levels (U/L) (FIG. 17B), CRP-5 (mg/L) (FIG. 17C), urea (mmol/L)
(FIG. 17D), CRE (.mu.mol/L) (FIG. 17E) and UA (.mu.mol/L) (FIG.
17F) in function of treatment. The graphs show representative
results (average of n=4.+-.S.E.). *P<0.05 was considered
significant based on student t-test.
[0049] FIG. 18 shows .sup.1H NMR spectra of Chitosan-PEG-TAT
(CS-O-PEG-CONH-TAT) (plot A), Chitosan-PEG-MGF (CS-O-PEG-CONH-MGF)
(plot B), Chitosan-PEG-P160 (CS-O-PEG-CONH-P160) (plot C) and
Chitosan-PEG-CP15 (CS-O-PEG-CONH-CP15) (plot D). The multiple peaks
of oxymethyl groups in PEG at .delta. 3.3 to 3.7 cover over the
signals of pyranose ring of chitosan. The multiple peaks at .delta.
6.0-9.0 belong to the peptide sequences respectively.
[0050] FIG. 19 shows transmission electron microscopy (TEM) image
of peptide-tagged pegylated chitosan nanoparticles complexing
siRNA. Magnification at 95 800.times.. Scale bar: 500 nm. The
average size of the nanoparticles ranged from 100 to 200 nm.
[0051] FIG. 20 shows gel retardation assay results to evaluate the
maximum gene (siRNA) loading efficiency of nanoparticles. Lane 1
represents a 10 bp DNA ladder used as a reference. Lane 2
represents 2 .mu.g/ml, lane 3 represents 4 .mu.g/ml, lane 4
represents 6 .mu.g/ml, lane 5 represents 8 .mu.g/ml and lane 6
represents 10 .mu.g/ml of siRNA dose complexed with the
nanoparticles. The results suggest that 8 .mu.g/ml is the optimal
amount of siRNA to be complexed with the nanoparticles.
[0052] FIGS. 21A-21E represent results of nanoparticles
accumulation in the tumor tissue after 4 hrs of dose
administration. FIGS. 21A-D show images of SW480 colon cancer
indicate the presence of scrambled biotinylated siRNA (0.5 mg/kg)
in the tumor tissue after intraperitoneal administration of the
treatment nanoparticle formulation. Animals were sacrificed after 4
hrs. Results are shown for the following nanoparticle formulations:
chitosan-PEG-CP15 (FIG. 21A), unmodified chitosan nanoparticles
(FIG. 21B), non-targeted biotin siRNA alone (FIG. 21C) and
untreated control cells (FIG. 21D). FIG. 21E shows image analysis
of the mean percent area stained in the tumor tissues of FIGS.
21A-21D. Results are shown as a percentage of biotin-NT siRNA
expression in function of treatment (chitosan-PEG-CP15: black bar,
unmodified chitosan nanoparticles: light gray bar, non-targeted
biotin siRNA alone: dark gray bar and untreated control cells. The
graph shows a representative result of the average of three random
sections measured per animal tissue, mean.+-.s.d. *p<0.05,
**p<0.01 were considered significant based on Tukey's post-hoc
analysis, when compared with other groups.
[0053] FIGS. 22A-22B represent analysis of biodistribution of siRNA
in various tissues 4 hrs after the administration of various
nanoparticles formulations. FIG. 22A shows histopathological
staining of heart, lungs, kidney, liver and spleen obtained from a
mouse xenografted with colon cancer and treated with various
nanoparticle formulations (administered at a dose of 0.5 mg/kg)
CS-P-CP15-NT siRNA: chitosan-PEG-CP15, CS-NT siRNA: unmodified
chitosan nanoparticles, NT-siRNA: non-targeting biotin-siRNA alone
and control as untreated. FIG. 22B shows image analysis of the mean
percent area stained in the tumor tissues shown in FIG. 22A.
Results are shown as mean percentage area stained for the presence
of the siRNA in function of the tissue studied and the nanoparticle
formulations administered (from left to right for each organs,
CS-P-CP15 NT siRNA, CS-NT siRNA, NT siRNA and control). The graphs
presents representative results of the average of two random
sections measured per animal tissue, mean.+-.s.d. *p<0.05,
**p<0.01 were considered significant based on Tukey's post-hoc
analysis, when compared with other groups.
[0054] FIG. 23 shows PLK1 mRNA expression in the tumor tissue after
intra-peritoneal administration with various nanoparticle
formulations. Results are shown as relative gene expression of PLK1
in tumor tissues in function of nanoparticle formulation:
CS-PEG-CP15-siRNA (.circle-solid.), CS-PEG-CP15 NT siRNA with
non-targeting biotin-siRNA (.box-solid.), PLK1 siRNA alone
(.tangle-solidup.) and saline (control ). The PLK1 gene expression
was compared among different groups after normalizing the GAPDH
levels among all the animals. A 50% PLK1 gene suppression was
observed in treated animals (p=0.031) as compared with untreated
controls. The graph shows a scatter dot plot with n=6 and
mean.+-.SE, *p<0.05 was considered significant based on Tukey's
post-hoc analysis, when compared with other groups.
[0055] FIG. 24 shows PLK1 protein expression in the tumor tissue
after intra-peritoneal administration with various nanoparticle
formulations. Results are shown as relative PLK1 protein expression
(obtained by western blot analysis from 100 .mu.g of total protein
extracted from tumor tissues of colon cancer and normalized against
the .beta.-actin protein levels) in function of nanoparticle
formulations: CS-PEG-CP15 with siRNA against PLK1 gene
(.circle-solid.), CS-PEG-CP15 with non-targeting biotin-siRNA
(.box-solid.), PLK1 siRNA alone (.tangle-solidup.) and saline
(control ). Reduction in PLK1 protein expression was observed with
treatment formulation as compared with untreated and controls. The
graph shows a scatter dot plot with n=6 and mean.+-.SE, *P<0.05
was considered significant based on Tukey's post-hoc analysis, when
compared with other groups.
[0056] FIGS. 25A-25B show results of serum analysis of mouse
treated with various nanoparticle formulations. In FIG. 25A,
AST/ALT ratios (U/L) are shown for mouse treated with CS-PEG-CP15
with siRNA against PLK1 gene (.circle-solid.), CS-PEG-CP15 with
non-targeting biotin-siRNA (.box-solid.), PLK1 siRNA alone
(.tangle-solidup.) and saline (control ). In FIG. 25B, C-reactive
protein levels (mg/dl) are shown for mouse treated with CS-PEG-CP15
with siRNA against PLK1 gene (.circle-solid.), CS-PEG-CP15 with
non-targeting biotin-siRNA (.box-solid.), PLK1 siRNA alone
(.tangle-solidup.) and saline (control ). No significant
differences between the treatment group and untreated controls were
observed. The graph shows a scatter dot plot with n=5 and
mean.+-.SE.
[0057] FIGS. 26A-26B show a CT scan of the neck before (FIG. 26A)
and after (FIG. 26B) the 10 day therapy cycle of Nora-PLK1 therapy
(intra-tumor), initiated on Sep. 27, 2014. The CT obtained on Oct.
13, 2014 shows a dramatic reduction of the tumor mass.
Volumetrically the tumor reduced from 29.66.times.41.66.times.46 mm
to 18.66.times.35.55.times.41 mm or from 56,839.2 m.sup.3 to
27,197.8 m.sup.3 (a 46% reduction).
[0058] FIGS. 27A-27D show photographic evidence of the reduction of
stage IV, squamous cell carcinoma (head and neck cancer) with the
Nora-PLK1 therapy (nanoparticle-siRNA), administered directly into
the tumor of a 55 years old, female patient. The dose administered
was 100 .mu.g of siRNA/day. FIG. 27A represents the state of tumor,
prior to therapy. FIG. 27B represents, reduction in the size of
tumor within first 3 weeks of the therapy. FIG. 27C represents,
complete reduction in the size of the tumor with presence of only
scar tissue. FIG. 27D represents, complete disappearance of tumor
mass and scarring tissue from the neck.
[0059] FIGS. 28A-28B show a chest CT scan of a 55 year old female
patient. FIG. 28A represents the tomogram from the Chest CT from
Sep. 27, 2014 and FIG. 28B represents the Chest x-ray from Nov. 26,
2014. As observed, a tumor mass is seen on the upper part of the
lower lobe of the right lung in FIG. 28A (white-dotted circle) that
is not present on the X-ray of FIG. 28B.
DETAILED DESCRIPTION
[0060] The present invention provides a delivery system for
specifically delivering agents to a tissue (or a group of tissues),
an organ (or a group of organs) or a cell type (or a group of cell
types). The delivery system is referred to as a non-viral
nanoparticles composed of a plurality of polymeric materials, each
comprising a hydrophilic linear polymer (such as PEG) covalently
associated at one end with a cross-linkable cationic polymer and at
the other end with a targeting/penetrating peptide. The
nanoparticles are not based on a viral system for delivering
agents. The cellular specificity of the delivery system can be used
to lower the dose of the agent used and, consequently, limit, if
any, the undesirable effects associated with the agent. The
nanoparticles can be used in vitro for research purposes, ex vivo
for cellular therapy applications as well as in vivo for therapy or
diagnostics. In some embodiments, the nanoparticles can be designed
to be more efficient at translocating across a biological membrane
and/or a cellular membrane (such as, for example, the cytoplasmic
membrane or the nuclear membrane).
Polymeric Material for Non-Viral Nanoparticles
[0061] The present invention provides a polymeric material that can
be used for the production of non-viral nanoparticles. The
polymeric material comprises at least three components, a linear
hydrophilic polymer (preferably a linear polyethylene glycol
polymer), a cross-linkable cationic polymer (preferably a chitosan
polymer) and a targeting/penetrating peptide. In the polymeric
material, the linear hydrophilic polymer is covalently associated,
at one end, to the cross-linkable cationic polymer and, at the
other end, to the targeting/penetrating peptide. The
targeting/penetrating peptide is indirectly covalently associated
with the cationic cross-linkable polymer (through the linear
hydrophilic polymer) but is not directly covalently associated with
the cross-linkable cationic polymer.
[0062] The components of the polymeric material can be directly and
covalently associated with one another. In such embodiment, there
is no linker between the cross-linkable cationic polymer and the
linear hydrophilic polymer nor between the linear hydrophilic
polymer and the targeting/penetrating peptide. However, in an
alternative embodiment, in some applications, a linker (either
between the cross-linkable cationic polymer and the linear
hydrophilic polymer and/or between the linear hydrophilic polymer
and the targeting/penetrating peptide) can be present. In some
instances, the linker can only be present between the
cross-linkable cationic polymer and the linear hydrophilic polymer
or between the linear hydrophilic polymer and the
targeting/penetrating peptide. In other instances, the linker can
be present between the cross-linkable cationic polymer and the
linear hydrophilic polymer and between the linear hydrophilic
polymer and the targeting/penetrating peptide. The linker between
the cross-linkable cationic polymer and the linear hydrophilic
polymer can be the same or can differ from the linker between the
linear hydrophilic polymer and the targeting/penetrating
peptide.
[0063] The cross-linkable cationic polymer of the polymer
biomaterial can be cross-linked to provide an internal core of
cross-linked cationic polymer. In addition, because of the positive
charge of the cross-linkable polymer, the internal core provided
upon cross-linking provides a micro-environment for a non-covalent
association with an anionic agent. In some embodiments, the
cross-linking between different polymeric material only occurs
between the cationic polymer moieties of from at least two
polymeric material. Otherwise stated, no or very little
cross-linking occurs between the cationic polymer and the linear
hydrophilic polymer or the targeting/penetrating peptide, no or
very little cross-linking occurs between the linear hydrophilic
polymer moieties of at least two distinct polymeric material, no or
very little cross-linking occurs between the linear hydrophilic
polymer and the targeting/penetrating peptide, and/or no or very
little cross-linking occurs between the targeting/penetrating
peptide moieties of at least two distinct polymeric material.
[0064] In a preferred embodiment, the cross-linkable cationic
polymer is chitosan. In some embodiments, chitosan can be modified
through its amine groups. However conjugating other compounds
through amines of chitosan can lead to the loss of its cationic
nature. The presence of free amine groups (e.g. a net positive
charge) on chitosan can help in complexing with negatively charged
agents (due to ionic interactions), can help in cellular uptake
and, once inside the endosomal cavity, can create a "proton sponge
effect" (e.g. a swelling behavior of chitosan observed when
encountering an acidic pH inside the cell's endosome). However, it
was observed that these free amine groups can be responsible for a
mild cellular toxicity. In the polymeric material described herein,
chitosan was chemically modified in order to preserve some of its
inherent cationic nature. In some embodiments, the amine groups of
the chitosan cause a surface charge (of the resulting nanoparticle
preferably having a size of less than about 100 nm) between 15 to
25 mV. In order to do so, the amine groups can be first be
protected then the chitosan can be conjugated the other functional
groups on chitosan with the hydrophilic linear polymer. The
expression "protecting amine groups" refer to the protection an
amine moiety with any suitable protecting groups. Examples of amine
protecting group can be found in Green et al., "Protective Groups
in Organic Chemistry", (Wiley, 4.sup.th ed. 2007) and Harrison et
al. "Compendium of Synthetic Organic Methods" (John Wiley and Sons,
1996).
[0065] In the polymeric material described herein, the degree of
deacetylation of the chitosan used can be 70%, 75%, 80%, 85% or
90%. In some embodiment, the degree of deacetylation of chitosan
can be 80%. In addition, in some embodiments, the chitosan used can
be a low (10-50 KDa), medium (50-200 KDa) or high (200-500 KDa)
molecular weight chitosan. In preferred embodiments, the chitosan
has an average molecular weight of the chitosan can be, for
example, about 100 000 Da.
[0066] Chitosan is unique among polysaccharides because it carries
amino groups which are positively charged in mildly acidic aqueous
solution (pH.ltoreq.5.5). As indicated above, the amine groups can
also be coupled to various chemical groups. In the polymeric
material described herein, the chitosan is modified (e.g.
covalently modified) to be attached (e.g. directly attached)
through a hydroxyl group to the linear hydrophilic polymer. In some
embodiments, various chitosan molecules also can be cross-linked
together via their amino groups.
[0067] In some embodiments, chitosan is not a water soluble variety
of chitosan. Water soluble chitosan are usually polymeric
oligomers, which are not efficient in complexing nucleic acid
molecules, such as siRNA. Due to the small size of the
water-soluble polymeric oligomers and the siRNA, the complexes
formed were shown not to be stable and were readily degraded by the
serum proteins. As such, if a water soluble chitosan is used, it
must be able to form a stable complex with the anionic agent, such
complex being capable of resisting degradation from serum
proteins.
[0068] In another preferred embodiment of the polymeric material,
the linear hydrophilic polymer can be a linear polyethylene glycol
(PEG) polymer. Throughout this application, the action of attaching
a linear PEG polymer to another entity is referred to as
"PEGylation" or "PEGylating" and a material having been attached to
a linear PEG can be qualified as "PEGylated". Once covalently
associated with the chitosan molecule, the PEG can reduce the
steric-hindrance of the nano-particle formed with the polymeric
material as well as reduce the inherent mild cellular cytotoxicity
associated with non-PEGylated chitosan. In preferred embodiments,
the PEG has an average molecular weight between about 2000 Da. In
some other specific embodiments, the PEG can also have an average
molecular weight of about 1 000 to 5 000 Da.
[0069] The third component of the polymeric material is a
targeting/penetrating peptide. As indicated herein, the
targeting/penetrating peptide has the ability to specifically bind
to a cell surface or to a cellular components. It thus provides
cell specificity and can also increase cellular uptake of the
polymeric material (as well as the non-viral particle produced from
the polymeric material). It can, for example, facilitate the
crossing of a cellular membrane (such as, for example, the nuclear
membrane). Alternatively, the targeting/penetrating peptide can be
to a cell surface or cell-associated surface receptor. In such
embodiments, the targeting/penetrating peptide can be a fragment or
derivative of a protein transduction domain or a fragment or
derivative of a growth factor. In this application, a single
targeting/penetrating peptide is covalently associated per linear
hydrophilic polymer. However, this does not limit the use single
peptide per nanoparticle formulation. For example, combination of
peptides can be used during the synthesis of the polymeric material
for making a population of different polymeric material, each
bearing a single peptide that can differ amongst the members of the
population. The use of a population of polymeric material can
further be used for the production of a multifunctional non-viral
nanoparticle.
[0070] In some embodiments, the targeting/penetrating peptide can
facilitate the translocation through a biological membrane. Such
targeting/penetrating peptide can be a TAT oligopeptide covalently
conjugated to the PEG polymer. TAT is a transcriptional activator
protein encoded by human immunodeficiency virus type 1 (HIV-1) and
is involved in the replication of the HIV virus. The basic domain
of TAT peptide comprises mainly of arginine and lysine amino acid
residues that have shown to play an important role in translocation
across biological membrane due to its strong cell-adherence and is
independent of receptors, temperature. The TAT peptide can be used
for facilitating the translocation of the nanoparticles across any
biological membrane. Nanoparticles comprising polymeric material
bearing the TAT peptide can also contain other polymeric material
bearing other targeting/penetrating peptides.
[0071] In other embodiments, the targeting/penetrating peptide can
be specific for neuronal cell. Such target peptide include, but are
not limited to MGF. MGF is a mechano growth factor. It is a splice
variant of IGF-1 (Insulin Growth Factor-1), whose receptors are
abundantly present on the surface of Purkinje cells in the
cerebellum. IGF-1 is a growth-promoting factor for Purkinje cell
development, particularly post-natal survival and dendritic growth.
IGF-1 is majorly involved in motor learning process and purkinje
cell synaptic plasticity. MGF has shown to promote motor neuron
survival but its neuroprotective action is independent of IGF-1.
MGF has been identified to have an affinity for myocardial and
neurological tissues and can be used as an effective
targeting/penetrating peptide for these tissues.
[0072] As also indicated herein, in some embodiments, the
targeting/penetrating peptide can be CP15. CP15 was identified by
the phage display technology. CP15 peptide has shown to be the most
effective peptide targeting colon tumor cells but not the normal
human intestinal epithelial human cells. CP15 peptide is used as a
ligand that will guide the nanoparticles to selectively target the
tumor tissue expressing receptors for CP15 peptide (colon
cancer).
[0073] In some embodiments, the targeting/penetrating peptide can
be P160. P160 was identified by phage display technology. P160
peptide has shown high affinity towards breast cancer and
neuroblastoma cells without affecting the normal endothelial cells.
P160 can be used as a ligand that will guide the nanoparticles to
selectively target the tumor tissue expressing receptors for P160
peptide (breast cancer).
Process for Producing the Polymeric Material
[0074] In order to produce the polymeric material, the hydrophilic
linear polymer can be first covalently associated with the
cross-linkable cationic polymer. Such covalent binding can be a
direct binding in which no linker is placed between the
cross-linkable cationic polymer and the hydrophilic linear polymer.
Alternatively, such covalent binding can be an indirect binding
where a linker is placed between the cross-linkable cationic
polymer and the hydrophilic linear polymer. Once the hydrophilic
linear polymer is covalently associated with the cross-linkable
cationic polymer, then the hydrophilic polymer is covalently
associated with the targeting/penetrating peptide. Such covalent
binding can be a direct binding in which no linker is placed
between the targeting/penetrating peptide and the hydrophilic
linear polymer. Alternatively, such covalent binding can be an
indirect binding where a linker is placed between the
targeting/penetrating peptide and the hydrophilic linear polymer.
As indicated above, there is no direct covalent association or
binding between the targeting/penetrating peptide and the
cross-linkable cationic polymer (even though some non-covalent
association may nevertheless be observed between the
targeting/penetrating peptide and the cross-linkable cationic
polymer).
[0075] In embodiments where the cross-linkable cationic polymer is
chitosan, some of (in some embodiments, the majority of) the amine
groups of the chitosan molecule can be protected prior to its
covalent association with the linear hydrophilic polymer. Such
protection can be conferred, for example, by phthaloylation. In
such embodiment, the amine groups of the chitosan molecule can be
de-protected after the covalent association of between the
hydrophilic linear polymer and the targeting/penetrating
peptide.
[0076] In some embodiments, a derivative of the linear hydrophilic
polymer can be used in the process. When the linear hydrophilic
polymer is PEG, one of the derivatives that can be used is
monomethyl ether polyethylene glycol (mPEG-OH). In such embodiment,
the mPEG can first be reacted (with succinic anhydride for example)
to form mPEG-COOH and then reacted (with thionyl chloride for
example) to form a mPEG-COCl. The mPEG-COCl (having a reactive
first end) can then be reacted with the cross-linkable cationic
polymer to form a PEGylated cross-linkable cationic polymer. Once
the PEGylated cross-linkable cationic polymer has been formed, the
other end of PEG containing methylether group (--OCH.sub.3) can
also be reacted to convert it to a hydroxyl group (with aluminium
chloride in ethanethiol for example) and then reacted to form an
activated PEG-COOH (with succinic anhydride for example). The
activated PEG-COOH (having a reactive second end) can then be
reacted with the at least one targeting/penetrating peptide to form
the complete polymeric material.
[0077] In an alternate embodiment to produce the polymeric
material, the PEG can be first covalently associated with the
targeting/penetrating peptide and then with the cross-linkable
cationic polymer. In such embodiment, the mPEG can first be reacted
(with succinic anhydride for example) to form mPEG-COOH and then
reacted (with thionyl chloride for example) to form a mPEG-COCl.
The mPEG-COCl (having a reactive first end) can then be reacted
with the targeting/penetrating peptide to form a PEGylated
targeting/penetrating peptide. Once the PEGylated
targeting/penetrating peptide has been formed, in an additional
embodiment, monomethyl ether polyethylene glycol (mPEG-OH) can also
be reacted to convert one of its methyl ether group to a hydroxyl
group (with aluminium chloride in ethanethiol for example) and then
reacted to form an activated PEG-COOH (with succinic anhydride for
example). The activated PEG-COOH (having a reactive second end) can
then be reacted with the cross-linkable cationic polymer to form
the polymeric material.
[0078] The person of ordinary skill in the art will understand that
the process described herein can be optimized and modified to
accommodate the various applications of the polymeric material and
should not be intended to be use to limit the scope of the
invention.
[0079] Non-Viral Nanoparticles
[0080] Due to the nature of the polymeric material used, the
nanoparticles described herein can be formed by simply admixing an
anionic agent with the positively charged cross-linkable polymer.
Through ionic interactions, the anionic agent complexes with the
cationic cross-linkable polymer. Upon the addition of a
cross-linker, the anionic agent becomes entrapped in the
cross-linked internal core formed by the cationic polymer. In the
non-viral nanoparticles, the anionic agent does not form a distinct
structure but is within the cross-linked mesh of the cationic
polymer. In some embodiments, the non-viral nanoparticles are shown
to exhibit low in vitro cytotoxicity. In embodiments, the
nanoparticles have been shown to cross physiological barrier such
as the blood-brain barrier (for example, when they have been
administered intra-nasally). In other embodiments, the
nanoparticles have been shown to be specifically delivered to a
tissue (for example, a solid tumor). In some embodiments, the
nanoparticles which comprise nucleic acid molecules as the
entrapped anionic agent have efficiently released the nucleic acid
into the cell's cytoplasm. In additional embodiments, the
nanoparticles can be designed for being targeted to the cell
nucleus (by using, for example, as one of the targeting/penetrating
peptide, a peptide that can facilitate the transfer through the
nuclear membrane).
[0081] As disclosed herein, a surface functionalized biocompatible
and biodegradable chitosan nanoparticles was developed and can be
used to increase the efficacy and stability of the nucleic
acid-based therapeutics and/or other therapeutic molecules. In
embodiments, the nanoparticles have been shown to provide sustained
targeted delivery. Without wishing to be bound to theory, FIG. 15
shows a schematic diagram of a proposed cellular uptake mechanism
of the non-viral nanoparticles. In this figure, the nanoparticles
are able to release the anionic agent at its targeted site. A
targeting ligand can be a specific peptide targeting, for example,
a cell-specific receptor. The use of a targeting/penetrating
peptide avoids the invasive delivery strategies such as
implantation of catheters, intracarotid infusions for therapeutic
delivery, surgeries and chemotherapies.
[0082] The nanoparticles-based drug-delivery system provided herein
is relatively inert and has low integration with physiological
system before reaching its targeted cell, organ or tissue. Some of
the nanoparticles can handle more payload, be modified with
appropriate ligands for specific cell targeting and be
administrated repeatedly without fretting about delivery induced
toxicity or immunogenicity. Some of the nanoparticles can be
designed for being less than about 100 nm in diameter and have a
positive surface charge. Some of the nanoparticles can overcome
anatomical, biophysical and physiological barriers, such as the
blood brain barrier. Some of the nanoparticles are able to
safe-guard the anionic agent against degradative enzymes before
reaching the targeted site. Some of the nanoparticles can be
designed to be target specific and/or can be administered
non-invasively.
[0083] In order to design the non-viral nanoparticles, various
properties of its constituents have been exploited. As indicated
above, one embodiment of the cross-linkable cationic polymer is
chitosan. Chitosan is a polycationic polymer that has been regarded
as a non-toxic, biodegradable and a biocompatible polysaccharide.
Due to the presence of primary amine groups, it is only soluble in
dilute acids. However, the solubility can be controlled by
specifically modifying the primary amine groups thereby making it
soluble in solvents like DMSO, DMF, pyridine, THF, etc. Chitosan of
low molecular weight has been shown to complex nucleic acids like
DNA at acidic pH and exhibit complex stability and integrity.
[0084] In addition, chitosan is also considered to be a
mucoadhesive agent which can reduce the clearance rate from the
nasal cavity. As such, in instances where the nanoparticles are
formulated for intranasal delivery (for example in intranasal
drops), it is believed that the nanoparticles are absorbed across
the nasal epithelia tissue, i.e. olfactory epithelium, and follow
olfactory/trigeminal neural pathways. In such embodiment, the pH at
which the nanoparticles can be formulated is 6.0, which is in
accordance with the pH range maintained in nasal cavity (pH 5.0 to
6.5). Chitosan has also been shown to interact with sialic acid
residues in the mucus to open the tight endothelial junctions. It
has further been shown to be used for intranasal delivery of
proteins.
[0085] As also indicated above, in some embodiments, the linear
hydrophilic polymer is polyethylene glycol (PEG). This polymer has
been extensively used in biomedical and pharmaceutical applications
because of its hydrophilic, non-toxic, non-antigenic and
non-immunogenic features. It is also a polymer which can be
chemically modified easily. In particularly, the use of
monofunctional PEG avoids nanoparticles to agglomerate and provides
resistance against enzyme degradation. In the context of the
present invention, the use of a bifunctional PEG (such as
mPEG-COOH) serves as a linker for conjugating two different
entities (a cross-linkable cationic polymer and a
targeting/penetrating peptide). Because it is neutral in charge and
reduces the steric-hindrance of the charged polymers, PEG enables
efficient conjugation of the two entities linked on its either
side.
[0086] The non-viral nanoparticles can comprise a single type of
polymeric material bearing the same linear hydrophilic polymer, the
same cross-linkable cationic polymer and the same
targeting/penetrating peptide. Alternatively, the non-viral
nanoparticles can also be made up of different polymeric materials.
For example, the non-viral nanoparticles can be made up of
polymeric material having the same linear hydrophilic polymer but
different cross-linkable cationic polymers and different
targeting/penetrating peptides. In another example, the non-viral
nanoparticles can be made up of polymeric material having the same
linear hydrophilic polymer and the same cross-linkable cationic
polymer but different targeting/penetrating peptides. In yet
another example, the non-viral nanoparticles can be made up of
polymeric material having different linear hydrophilic polymers but
the same cross-linkable cationic polymer and the same
targeting/penetrating peptide. In still another example, the
non-viral nanoparticles can be made up of polymeric material having
the same linear hydrophilic polymer and the same different
targeting/penetrating peptide but different cross-linkable cationic
polymers. In yet another example, the non-viral nanoparticles can
be made up of polymeric material having the same cross-linkable
cationic polymer but different linear hydrophilic polymers and
different targeting/penetrating peptides. In still another example,
the non-viral nanoparticles can be made up of polymeric material
having the same targeting/penetrating peptide, but different linear
hydrophilic polymers and different cross-linkable cationic
polymers.
[0087] In a specific embodiment, the non-viral nanoparticle is
composed of a plurality of polymeric materials. One of the
polymeric material can comprise a first polymeric material having a
first targeting/penetrating peptide that can bind to a cell surface
or cell-associated receptor (for example, the MGF, CP15 or P160
targeting/penetrating peptide). Another polymeric material can
comprise a second polymeric material having a second peptide that
can facilitate the translocation of the non-viral particle across a
cellular membrane (for example the TAT penetrating peptide). Such
nanoparticles is thus composed of at least two different
targeting/penetrating peptides, one for facilitating the
translocation across a biological membrane and another one for
providing cellular or tissue-specificity.
[0088] The non-viral nanoparticles comprise an anionic agent. The
agent should bear a net negative charge in order to complex easily
with the cationic polymer. The agent can be used for research
purposes, therapeutic purposes and/or diagnostic purposes.
[0089] In some embodiment, the anionic agent is a nucleotide-based
agent for lowering or inhibiting gene expression and/or protein
expression. Such agents include, but are not limited to antisense
oligonucleotide, triplex oligonucleotide, miRNA, siRNA, shRNA and
ribozyme. In some embodiments, a single type of anionic agent is
present in the non-viral nanoparticles. In other embodiments, more
than one types of anionic agent is present in the non-viral
nanoparticles.
[0090] An antisense oligonucleotide is wholly or partially
complementary to, and can hybridize with, a target nucleic acid
(either DNA or RNA). For example, an antisense nucleic acid or
oligonucleotide comprising about 15 to 35 nucleotides spanning the
coding/non-coding sequence of a gene or its corresponding
transcript whose expression is to be inhibited. In another
embodiment, the antisense nucleic acid is wholly or partially
complementary to, and can hybridize with, a target nucleic acid. As
non-limiting examples, antisense oligonucleotides may be targeted
to hybridize to the following regions: mRNA cap region, translation
initiation site, translational termination site, transcription
initiation site, transcription termination site, polyadenylation
signal, 3' untranslated region, 5' untranslated region, 5' coding
region, mid-coding region, 3' coding region, DNA replication
initiation and elongation sites.
[0091] Triplex oligonucleotides, much like antisense
oligonucleotides can inhibit transcription and/or expression of a
target gene or its transcripts. Triplex oligonucleotides are
constructed using the base-pairing rules of triple helix formation
and the nucleotide sequence of the target genes. They are usually
between 10 and 40 or between 15 to 25 nucleotides-long.
[0092] RNA interference (RNAi) is a post-transcriptional gene
silencing process that is induced by a miRNA or a dsRNA (a small
interfering RNA (siRNA) or small hairpin RNA (shRNA)) and has been
used to modulate gene expression. While the invention is not
limited to a particular mode of action, RNAi may involve
degradation of messenger RNA by an RNA induced silencing complex
(RISC), preventing translation of the transcribed targeted mRNA.
Oligonucleotides that can mediate RNAi are generally at least 10
nucleotides long. Because manipulation of RNA may be complex, RNAi
can be provided with a deoxyribonucleic acid (DNA) compositions
encoding small interfering RNA (siRNA) molecules, or intermediate
siRNA molecules (such as shRNA), comprising one strand of an
siRNA.
[0093] Small interfering RNA or siRNA includes any nucleic acid
molecule capable of mediating RNA interference "RNAi" or gene
silencing. For example, siRNA of the present invention can be
double stranded RNA molecules from about 10 to about 30 nucleotides
long that are named for their ability to specifically interfere
with protein expression. In one embodiment, siRNAs of the present
invention are 12 to 28 nucleotides long, more preferably 15 to 25
nucleotides long, even more preferably 19 to 23 nucleotides long
and most preferably 21 to 23 nucleotides long. Therefore preferred
siRNA of the present invention are 12, 13, 14, 15, 16, 17, 18, 19,
20, 21, 22, 23, 24, 25, 26, 27, 28 nucleotides in length. As used
herein, siRNA molecules need not to be limited to those molecules
containing only RNA, but further encompass chemically modified
nucleotides and non-nucleotides. An siRNA molecule can be assembled
from two nucleic acid fragments wherein one fragment comprises the
sense region and the second fragment comprises the antisense region
of siRNA molecule. The sense region and antisense region can also
be covalently connected via a linker molecule. The linker molecule
can be a polynucleotide linker or a non-polynucleotide linker. In
an embodiment, the siRNA can target the PSN1 gene or the PLK1
gene.
[0094] A ribozyme (from ribonucleic acid enzyme, also called RNA
enzyme or catalytic RNA) is an RNA molecule that catalyzes a
chemical reaction. Some ribozymes may play an important role as
therapeutic agents, as enzymes which target defined RNA sequences.
Ribozymes can be genetically engineered to specifically cleave a
transcript of a gene from a candidate region that is being
upregulated with the disease.
[0095] The oligonucleotides described herein can be
naturally-occurring species or synthetic species formed from
naturally-occurring subunits or their close homologs. The term may
also refer to moieties that function similarly to oligonucleotides,
but have non-naturally-occurring portions. Thus, oligonucleotides
may have altered sugar moieties or inter-sugar linkages. Exemplary
among these are phosphorothioate and other sulfur containing
species which are known in the art. In some embodiments, at least
one of the phosphodiester bonds of the oligonucleotide has been
substituted with a structure that functions to enhance the ability
of the compositions to penetrate into the region of cells where the
RNA whose activity is to be modulated is located. It is preferred
that such substitutions comprise phosphorothioate bonds, methyl
phosphonate bonds, or short chain alkyl or cycloalkyl structures.
In accordance with other embodiments, the phosphodiester bonds are
substituted with structures which are, at once, substantially
non-ionic and non-chiral, or with structures which are chiral and
enantiomerically specific. Persons of ordinary skill in the art
will be able to select other linkages for use in the practice of
the invention. Oligonucleotides may also include species that
include at least some modified base forms. Thus, purines and
pyrimidines other than those normally found in nature may be so
employed. Similarly, modifications on the furanosyl portions of the
nucleotide subunits may also be affected, as long as the essential
tenets of this invention are adhered to. Examples of such
modifications are 2'-O-alkyl- and 2'-halogen-substituted
nucleotides. Some non-limiting examples of modifications at the 2'
position of sugar moieties which are useful in the present
invention include OH, SH, SCH.sub.3, F, OCH.sub.3, OCN,
O(CH.sub.2), NH.sub.2 and O (CH.sub.2).sub.nCH.sub.3, where n is
from 1 to about 10. Such oligonucleotides are functionally
interchangeable with natural oligonucleotides or synthesized
oligonucleotides, which have one or more differences from the
natural structure.
[0096] In other embodiments, the anionic agent is a
nucleotide-based agent for promoting or increasing gene expression
and/or protein expression. Such agents include, but are not limited
to expression vectors. In some embodiments, a single type of
anionic agent is present in the non-viral nanoparticles. In other
embodiments, more than one types of anionic agent is present in the
non-viral nanoparticles.
[0097] Expression vectors can be derived from viruses
(retroviruses, adenovirus, adeno-associated viruses, herpes,
lentiviruses and/or vaccinia viruses) or from various bacterial
plasmids may be used for delivery of nucleotide sequences to the
targeted organ, tissue or cell population. Methods which are well
known to those skilled in the art can be used to construct
recombinant vectors which will express nucleic acid sequence that
is complementary to a specific (or a combination) of nucleic acid
sequence. Viral vector delivery systems include DNA and RNA
viruses, which have either episomal or integrated genomes after
delivery to the cell.
[0098] The non-viral nanoparticles of the invention are small
enough to translocate across a biological membrane. In some
embodiment the diameter of the nanoparticles is between about 5 to
about 100 nm. In other embodiments, the diameter of the
nanoparticles are between about 50 to about 300 nm. In an
embodiment, the non-viral nanoparticles have a diameter of less
than 5 nm. In another embodiment, the non-viral nanoparticles have
a diameter between 5 and 10 nm. In a further embodiment, the
non-viral nanoparticles have a diameter between 100 and 200 nm.
Process for the Production of the Non-Viral Nanoparticles
[0099] In some embodiments, the non-viral nanoparticles can be
obtained in a one-step fashion. A solution comprising both the
anionic agent and the cross-linker is added to a plurality of
polymeric materials (identical or different). In an embodiment,
when the anionic agent is a siRNA, the ratio between the siRNA and
the polymeric materials is at least 2 .mu.g/ml, at least 4
.mu.g/ml, at least 6 .mu.g/ml, at least 8 .mu.g/ml or at least 10
.mu.g/ml of the siRNA for each 0.5 mg/ml of the polymeric material
solution. In a preferred embodiment, the ratio between the siRNA
and the polymeric materials is about 2 .mu.g/ml of siRNA to be
complexed 0.5 mg/ml of polymeric material solution (with average
molecular weight of chitosan of 10 KDa) to form nanoparticles in
range 5 to 100 nm. In another preferred embodiment, the ratio
between the siRNA and the polymeric material is about 8 .mu.g/ml of
the siRNA for each 0.5 mg/ml of the polymeric material solution
(with average molecular weight of chitosan 50 to 190 KDa) to form
nanoparticles in range 50 to 300 nm. The process for making a
non-viral nanoparticle includes an anionic cross linker, that can
be sodium tripolyphosphate (TPP). The anionic agent (siRNA) along
with the anionic cross-linker (TPP) are admixed and then added
drop-wise to a cationic polymeric solution in conditions favoring
the formation of nanoparticles through ionic interactions. As
indicated herein, cross-linking is limited to the cross-linkable
cationic polymer moiety of the polymeric material. Cross-linking is
not observed between the other components of the polymeric material
or the anionic agent. The resulting nanoparticles will have an
internal core (in which the anionic agent is entrapped in the
cross-linked cationic polymer) having protrusions of hydrophilic
linear polymers being covalently associated with the
targeting/penetrating peptide. The size of the resulting
nanoparticles are in the nanometers range. The shape of the
resulting nanoparticules is spherical. In some embodiments, the
relative diameter of the non-viral nanoparticle is between about 5
to about 100 nm. In other embodiments, the relative diameter of the
non-viral nanoparticle is between about 50 to about 300 nm.
[0100] Exemplary cross-linkers include, but are not limited to
small chemical entities having either amine reactive groups,
carboxyl reactive groups, aldehyde reactive groups, ketone reactive
groups, hydroxyl reactive groups, thiol reactive groups and
hydrazide reactive groups. Examples of linkers can be found in Greg
T. Hermanson, "Bioconjugate techniques" (Elsevier Inc. 2008). More
specifically, the cross-linkers include, but are not limited to
zero-length crosslinkers (such as, for example, carbodiimides (EDC,
EDC and sulfo-NHS, CMC, DCC, DIC), Woodward's Reagent K, N,
N-carbonyldiimidazole and Schiff Base Formation and Reductive
Amination), homobifunctional crosslinkers (such as, for example,
homobifunctional NHS esters (DSP and DTSSP, DSS and BS, DST and
Sulfo-DST, BSOCOES and Sulfo-BSOCOES, EGS and Sulfo-EGS, DSG, DSC),
homobifunctional imidoesters (such as, for example DMA, DMP, DMS,
DTBP), homobifunctional sulfhydryl-reactive crosslinkers (DPDPB,
BMH, difluorobenzene derivatives, DFDNB, DFDNPS), homobifunctional
photoreactive crosslinkers (such as, for example, BASED),
homobifunctional aldehydes (such as, for example, formaldehyde,
glutaraldehyde), bis-epoxides (such as, for example, 1,4-butanediol
diglycidyl ether), homobifunctional hydrazides (such as, for
example, adipic acid dihydrazide), carbohydrazide (such as, for
example, bis-diazonium derivatives, o-Tolidine, diazotized,
bis-diazotized benzidine) and bis-alkyl halides),
heterobifunctional crosslinkers (amine-reactive and
sulfhydryl-reactive crosslinkers (such as, for example, SPDP,
LC-SPDP, and Sulfo-LC-SPDP; SMPT and Sulfo-LC-SMPT; SMCC and
Sulfo-SMCC; MBS and Sulfo-MBS; SIAB and Sulfo-SIAB; SMPB and
Sulfo-SMPB; GMBS and Sulfo-GMBS; SIAX and SIAXX; SIAC and SIACX;
NPIA), carbonyl-reactive and sulfhydryl-reactive crosslinkers (such
as, for example, MPBH, M2C2H, PDPH), amine-reactive and
photoreactive crosslinkers (such as, for example, NHS-ASA,
sulfo-NHS-ASA, and sulfo-NHS-LC-ASA; SASD; HSAB and Sulfo-HSAB;
SANPAH and Sulfo-SANPAH; ANB-NOS; SAND; SADP and Sulfo-SADP;
Sulfo-SAPB; SAED; Sulfo-SAMCA; p-Nitrophenyl Diazopyruvate;
PNP-DTP), sulfhydryl-reactive and photoreactive crosslinkers (such
as, for example, ASIB, APDP, Benzophenone-4-iodoacetamide,
Benzophenone-4-maleimide), carbonyl-Reactive and Photoreactive
Crosslinkers (such as, for example, ABH), carboxylate-reactive and
photoreactive crosslinkers (such as, for example, ASBA),
arginine-reactive and photoreactive crosslinkers (such as, for
example, APG)), or trifunctional crosslinkers
(4-Azido-2-nitrophenylbiocytin-4-nitrophenyl ester, Sulfo-SBED,
MTS-ATF-Biotin and MTS-ATF-LC-Biotin, Hydroxymethyl Phosphine
Derivatives).
[0101] In some specific applications, it is sufficient (and
sometimes even necessary) to provide identical polymeric material
for making the nanoparticles. However, in other applications, it is
preferably to have different polymeric material in the
nanoparticle. In one specific example, it is contemplated that two
different polymeric material be provided, each one only differing
in the type of peptide that they bear. For example, the two
different polymeric material can each bear either a
targeting/penetrating peptide that can bind to a cell surface or
cell-associated receptor (such as MGF, CP-15 or P160 for example)
or a targeting/penetrating peptide that can facilitate the
translocation of the non-viral particle across a cellular membrane
(such as TAT for example).
Use of the Non-Viral Nanoparticles
[0102] The non-viral nanoparticles described herein can be used to
specifically deliver an anionic agent to a cell. In order to do so,
an appropriate amount of the non-viral nanoparticle is contacted
with the cell and incubated in conditions appropriated to allow the
entry of the nanoparticles into the cell. In some embodiments, such
contact between the nanoparticles and the cell occurs in vitro.
[0103] In other embodiments, the cell remains in vitro after the
contact (and following the entry of the nanoparticles). However, in
some embodiments, the contacted cell can be introduced into an
individual in need thereof. Such embodiments may be useful for
providing ex vivo gene therapy to the individual. For example,
cells explanted from an individual patient (e.g., lymphocytes, bone
marrow aspirates, and tissue biopsy) or universal donor
hematopoietic stem cells, followed by re-implantation of the cells
into the individual, usually after selection for cells which have
incorporated the nanoparticles. In one embodiment, stem cells are
used in ex vivo procedures for cell transfection and gene therapy.
The advantage to using stem cells is that they can be
differentiated into other cell types in vitro, or can be introduced
into a mammal (such as the donor of the cells) where they will
engraft at an appropriate location (such as in the bone
marrow).
[0104] In alternative embodiments, the non-viral nanoparticle is
formulated for administration prior to being administered to the
individual. Such formulation can provide a systemic administration
(intravenous, intraperitoneal, intramuscular, subdermal, or
intracranial infusion) or a topical administration (cutaneous,
intranasal, mucosal, etc.). Although more than one route can be
used to administer the nanoparticles, a particular route can often
provide a more immediate and more effective reaction than another
route. The nanoparticles of the present invention may be
administered, either orally or parenterally, systemically or
locally. For example, intravenous injection such as drip infusion,
intramuscular injection, intraperitoneal injection, subcutaneous
injection, suppositories, intestinal lavage, oral enteric coated
tablets, and the like can be selected, and the method of
administration may be chosen, as appropriate, depending on the age
and the conditions of the patient. Since some of the nanoparticles
are designed to cross efficiently biological barrier (such as the
BBB, the mucosa or/and the skin), the nanoparticles can be
formulated for contacting such biological barrier.
[0105] In some embodiments, the nanoparticles are used for
delivering a therapeutically effective amount of the anionic agent
to the brain of an individual in need thereof. The nanoparticles
can be formulated as nasal drops for intranasal administration.
Such embodiments includes the prevention, treatment and/or
alleviation of symptoms associated to a neurodegenerative disease
or a brain cancer. Exemplary embodiments of the neurodegenerative
diseases include but are not limited to spinocerebellar ataxia,
Huntington's disease, Parkinson's disease, Alzheimer's disease,
dementia and/or amyotrophic lateral sclerosis. Also contemplated
herein are non-viral nanoparticles for the delivery of agents to
the brain that can also be used for the prevention, treatment
and/or alleviation of symptoms associated to a neurodegenerative
disease or a brain cancer
[0106] In other embodiments, the nanoparticles can be used for
delivering a therapeutically effective amount of an anionic agent
to a cancer cell (such as a carcinoma cell). The nanoparticles can
be formulated for intravenous/intraperitoneal/intratumor
administration prior to being administered to the individual. The
nanoparticles can be used for the prevention, treatment and/or
alleviations of symptoms associated with a proliferative disease,
such as cancer (e.g., a colon cancer).
[0107] Also contemplated herein are non-viral nanoparticles for the
delivery of agents to the brain that can also be used for the
prevention, treatment and/or alleviation of symptoms associated to
a cancer. The nanoparticles can be administered alone or in
conjunction with other anti-neoplastic compounds. The use of the
nanoparticles can also be combined with radiation therapy.
[0108] The present invention will be more readily understood by
referring to the following examples which are given to illustrate
the invention rather than to limit its scope.
EXAMPLE I
Production and Characterization of the Nanoparticles
[0109] Material and methods. Low Molecular weight (LMW) chitosan
(M.W. 10 KDa) was obtained from Wako (Richmond, Va., USA), having a
viscosity of 5.about.20 cP (at room temperature) and degree of
deacetylation of 80.0%. Polyethylene glycol monomethyl ether
(mPEG): medium molecular weight (2 000 Da), phthalic anhydride,
pyridine, toluene, hydrazine monohydrate, succinic anhydride,
ethanethiol, aluminium chloride, sodium tripolyphosphate (TPP),
agarose, ethidium bromide (10 mg/ml) and glacial acetic acid of
analytical grade were obtained from Sigma (Oakville, ON, Canada).
Anhydrous N,N-Dimethylformamide (DMF),
1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride) (EDC),
4-(Dimethylamino) pyridine (DMAP), sodium hydroxide (NaOH),
methanol, diethyl ether, chloroform and concentrated hydrochloric
acid were obtained from Thermo Fisher Scientific (Ottawa, ON,
Canada). Thionyl chloride (SoCl.sub.2) was obtained from VWR
(Mississauga, ON, Canada). The peptides, TAT peptide
(NH.sub.2-RKKRRQRRR-NH.sub.2) (SEQ ID NO: 4) M.W. 1339.40, and MGF
peptide (NH.sub.2-YQPPSTNKNTKSQRRKGSTFEEHK-NH.sub.2) (SEQ ID NO: 1)
M.W. 2848.14, were synthesized by Sheldon Biotech, McGill
University. Mouse Neuro2a cells and EMEM media were obtained from
Cedarlane laboratories (Burlington, Ontario, Canada). MTS
cytotoxicity assay kit was obtained from Promega (Madison, Wis.,
USA). Subcloning DH5-alpha competent cells and Lipofectamine 2000,
TrackIt 10-bp DNA ladder (0.5 .mu.g/.mu.l), Pre-cast Nupage 4-12%
Bis-Tris gels, Nupage MES running buffer, Nupage transfer buffer,
Nupage LDS sample buffer (4.times.), nitrocellulose membrane 0.45
.mu.m, blotting filter paper and magic mark (1 Kb) protein ladder
were obtained from Invitrogen (Burlington, ON, Canada). Orange dye
solution (6.times.) was obtained from Fermentas (Burlington, ON,
Canada). pEGFP.sup.ataxin1 (E3-82Q) plasmid was obtained from Dr.
Zoghbi Huda from Baylor College of Medicine, Houston, USA. Plasmid
purification Maxi kit is from Qiagen (Mississauga, Ontario,
Canada). Primary antibodies: Ataxin-1(L-19) goat polyclonal
antibody, Actin (l-19) goat polyclonal antibody and
(HRP)-conjugated donkey anti-goat IgG antibody were procured from
Santacruz Biotechnology (Santa Cruz, Calif., USA). Ataxin-1 siRNA
(h) was procured from Santacruz Biotechnology (Santa Cruz, Calif.,
USA). SiGLO red (tagged with Cy3), was obtained from Thermo Fisher
Scientific (Ottawa, ON, Canada). Scrambled/Non targeting (NT) siRNA
tagged with biotin was obtained from Dharmacon (Lafayette, Colo.,
USA).
[0110] Commercially available low molecular weight chitosan (CS)
with an average molecular weight of 10 000 Da and with degree of
deacetylation (DA) of 80% (1.00 g) was added to a solution of
phthalic anhydride (PH) (2.76 g) in 20 ml of N,N-Dimethylformamide
(DMF) in order to protect the amine groups of chitosan. The mixture
was stirred for 8 hours at 110.degree. C. under nitrogen atmosphere
(Kurita et al., 2002). The resultant product (c) in FIG. 1 was
cooled to room temperature and precipitated in ice cold water. The
precipitate was filtered, washed with methanol overnight and vacuum
dried. This is an important step in carrying forward any further
chemical reactions with chitosan, as chitosan being soluble only in
acidic conditions becomes soluble in most organic solvents such as
DMSO, DMF, THF, pyridine etc. after protection with phthalic
anhydride.
[0111] Phthaloylation of the chitosan polymer. .sup.13C NMR data
confirms successful N-phthaloylation of the product. Product (c) in
FIG. 1 shows the schematic representation of the N-phthaloyl
chitosan. The Total Suppression of Side bands (TOSS) mode exhibit
peaks at 124.62 ppm, 131.70 ppm, 134.42 ppm corresponding to
phenylene, and 169.66 ppm and carbonyl group of phthaloyl group and
the TOSS-dipolar dephasing (TOSDL) mode confirms the absence of CH
and CH.sub.2 peaks due to their short relaxation time and presence
of only two peaks 131.63 ppm and 169.89 ppm assigned to C 1, 2 and
carbonyl of phthaloyl group respectively. The IR data shows
distinct sharp peaks at 1774 cm.sup.-1 and 1702 cm.sup.-1
corresponding to imide of phthaloyl group in FIG. 2. The phthaloyl
chitosan prepared by this method becomes gel-like when precipitated
in water, which supports the data shown by Kurita, of formation of
a uniform structure of phthaloylated chitosan (Kurita et al.,
2002).
[0112] Pegylation of chitosan: Synthesis of mPEG-COCl. To 10%
solution of monomethyl ether PEG (OH-PEG-OCH.sub.3) (M.W. 2 000) in
toluene was converted to carboxylate-terminated PEG
(COOH-PEG-OCH.sub.3) by reacting it with four molar excess of
succinic anhydride (dissolved in pyridine). The above reaction was
set up under anhydrous conditions and refluxed for 6 hours. The
product was precipitated with ether, filtered and again
re-precipitated twice with chloroform and diethyl ether and finally
vacuum dried. The final product obtained (a), is shown in FIG.
1.
[0113] FIG. 3 represents the IR spectra that shows the presence of
carboxylic peak at 1732 cm.sup.-1, confirming the presence of
(--C.dbd.O) carbonyl groups on PEG. To mPEG-COOH obtained in the
previous reaction, 2 fold molar excess of SOCl.sub.2 was added
under N.sub.2 atmosphere to form PEG acyl chloride
(COCl-PEG-OCH.sub.3) (product b in FIG. 1). The reaction was
refluxed to boil for 6 hours, followed by degassing to remove
excess SO.sub.2 and thionyl chloride.
[0114] Pegylation of chitosan: Synthesis of CSPH-O-mPEG (PEGylating
phthaloyl chitosan). This product (b) PEG acyl chloride
(COCl-PEG-OCH.sub.3) obtained in previous reaction is used as an
active intermediate to be further conjugated to hydroxyls of
2-N-phthaloyl chitosan (Product c). For this reaction, phthaloyl
chitosan (CSPH) (300 mg), product (c), was soaked in pyridine
solution overnight and added to mPEG acylchloride (10 g), product
(b), in toluene. The reaction was stirred for 2 hours at room
temperature and then refluxed to boil for 24 hours (Lin et al.,
2007). The resultant product was allowed to cool at room
temperature, precipitated in methanol and vacuum dried to yield
product (d) in FIG. 1.
[0115] The FTIR spectra in FIG. 4A shows PEG grafted phthaloylated
chitosan with characteristic peaks at 2873 cm.sup.-1 (C--H
stretching), 1068 cm.sup.-1 (C--O stretching), 1491 cm.sup.-1, 1451
cm.sup.-1 and 1254 cm.sup.-1 belong to PEG. Also the reduction in
hydroxyl peaks of chitosan at 3200-3500 cm.sup.-1, indicates the
grafting of PEG onto chitosan forming 2-N-phthaloyl
chitosan-O-mPEG, product (d) in FIG. 1.
[0116] Tagging a peptide to a pegylated chitosan polymer. To
conjugate peptide onto PEGylated phthaloyl chitosan, the methoxy
group of PEG was converted to hydroxyl group by following the
procedure as mentioned in Lin et al. (2007). In brief equi-molar
ratios of CS-mPEG and aluminium chloride were reacted together for
12 hours at room temperature in 20 ml of ethanethiol. The reaction
mix was diluted with water, acidified with 10% HCl, filtered and
extracted thrice with dichloromethane to yield product (e) in FIG.
1.
[0117] The hydroxyl groups formed (CSPH-PEG-OH) were further
converted to carboxyl groups by reacting it with 4 molar excess of
succinic anhydride in toluene at 100.degree. C. for 12 hours. The
product obtained as (f) in FIG. 1 was cooled at room temperature,
precipitated with methanol, filtered and vacuum dried. The
carboxylic terminal groups of this product (f) i.e. CS-PEG-COOH (12
mM) was conjugated to the equimolar ratio of peptide-NH.sub.2 (12
mM) in the presence of EDC (15.5 mM) and DMAP (1.2 mM) in 1 ml of
DMF for 24 hrs. This reaction mix was dialyzed against deionized
water for 2 days, using a dialysis tube with molecular weight
cut-off of 3 500 Da. The resultant product (g) was obtained as
shown in FIG. 1.
[0118] The IR spectra in FIG. 4B shows the appearance of a new peak
at 1659 cm.sup.-1 that shows the presence of amide bonds and
confirms the conjugation of peptide onto the polymer forming
2-N-phthaloyl chitosan-O-PEG-CONH-peptide, product (g) in FIG.
4.
[0119] Deprotection of chitosan polymer-CS-PEG-peptide (TAT/MGF).
As a last step of the synthesis, the amine groups of chitosan were
deprotected using 5% hydrazine monohydrate in DMF. The reaction was
carried out at 100.degree. C. for an hour under inert conditions.
The mixture was allowed to cool at room temperature and was
dialyzed against deionized water for 2 days, using a dialysis tube
with molecular weight cut-off of 3 500 Da. The sample obtained
after dialysis was vacuum dried and marked as product (h) in FIG.
1. Hydrazine monohydrate being basic in nature causes
destabilization of the phthaloyl moiety by creating an excess
alkaline condition of pH>12.
[0120] The removal of phthaloyl group from the polymer was
confirmed by FTIR, where the absence of peaks at 1775 cm.sup.-1 and
1710 cm.sup.-1 confirmed the complete dissociation of phthalimido
group from chitosan (FIG. 4C). It was observed that the reaction
conditions followed in the scheme at certain steps had no
deleterious effects on any of the previous bonds formed. The peak
representing the presence of amides due to the peptide shifts from
1659 cm.sup.-1 to 1644 cm.sup.-1 and the peak at 1582 cm.sup.1
(amide II) belongs to the chitosan. The peaks at 2918 cm.sup.-1 and
1061 cm.sup.-1 refers to the presence of (CH.sub.2) groups and
(C--O stretch) of PEG respectively as observed in FIG. 4c. The
final product was dialyzed for 2 days against deionized water in
order to get rid of any impurity or byproduct left during the
reaction.
[0121] Polymer characterization. The polymer CS-PEG-peptide was
characterized and analyzed at every step of its derivation through
FTIR (Perkin Elmer Spectrum BX), .sup.13C (Varian 300 MHz broadband
NMR) and .sup.1H (Mercury 400 and 500 MHz NMR) and Transmission
Electron Microscopy (Philips EM410 TEM).
[0122] FIG. 5 represents the .sup.1H NMR spectra of 2-N-phthlaoyl
chitosan (FIG. 5A), 2-N-phthlaoyl chitosan-O-mPEG (FIG. 5B),
2-N-phthaloyl chitosan-O-PEG-CONH-TAT (FIG. 5C) and
chitosan-O-PEG-CONH-TAT (FIG. 5D). The chemical shift at .delta.
7.78 belongs to the aromatic protons of the phthaloyl moiety, which
is present in the spectra of 2-N-Phthlaoyl chitosan-O-mPEG,
2-N-phthaloyl chitosan-O-PEG-CONH-TAT but disappeared in
chitosan-O-PEG-CONH-TAT spectrum. The multiple peaks of oxymethyl
groups in PEG at .delta. 3.3 to 3.7 cover over the signals of
pyranose ring of chitosan in all the three spectres. The weak and
broad peak at .delta. 4.3-4.5 were from the protons of
--NH--CH(CH.sub.2)--CO-- in TAT peptide as observed in spectrum
2-N-phthaloyl chitosan-O-PEG-CONH-TAT. The peak at .delta. 2.7-2.8
came from the protons of --CH.sub.2--NH--NH--NH.sub.2 in arginine
and the weak and multiple peaks at .delta. 1.3-1.7 came from the
--CH.sub.2--CH.sub.2--CH.sub.2--NH--NH--NH.sub.2 in arginine as
observed in spectra 2-N-phthaloyl chitosan-O-PEG-CONH-TAT and
chitosan-O-PEG-CONH-TAT. The multiple peaks at .delta. 7.0-8.0
belong to the amines in TAT peptide sequence (FIG. 5E).
[0123] Preparation of siRNA complexes with CS-PEG-PEPTIDE (TAT/MGF)
nanoparticles. FIG. 6 represents TEM images of the polymer
chitosan-O-PEG-CONH-peptide dissolved in dilute acetic acid at pH 6
at a concentration of 0.5 mg/ml, which was sonicated for 10 minutes
before being observed under the transmission electron microscope
(TEM). FIG. 6A shows the TEM image of the polymer PEGylated
chitosan polymer at magnification 53 800.times. and FIG. 6B is the
peptide tagged PEGylated chitosan polymer at magnification 53
800.times.. The spherical shape of the particles formed is
attributed to the conjugation of PEG on chitosan. FIG. 6C shows
effect of acidic pH (5.5) on peptide tagged PEGylated chitosan
polymer at magnification 70 700.times. formed before complexing
siRNA. The polymer appeared to disperse and disintegrate at acidic
pH.
[0124] To form siRNA complexed nanoparticles,
scrambled/non-targeting (NT) siRNA (siGLO-red) was mixed with TPP
at pH 3, as described previously (Malhotra et al., 2009) and then
added drop-wise to the polymer solution of chitosan-PEG-peptide at
a concentration 0.5 mg/ml. Final concentration of siRNA used was 2
.mu.g/ml. The solution was stirred for an hour at room temperature.
The nanoparticles obtained were viewed under TEM.
[0125] FIG. 6D shows nanoparticles formed with the modified
Chitosan-PEG-peptide polymer, without siRNA, using TPP as a
crosslinker. The nanoparticles range in the size of 50-100 nm. FIG.
6E and 6F represent nanoparticles formed with unmodified chitosan
complexing siRNA and Chitosan-PEG-peptide polymer complexing siRNA
respectively. On encapsulating siRNA, the nanoparticles obtained
from chitosan-PEG-peptide polymer were smaller than 20 nm and
appeared more spherical and monodispersed, (FIG. 6F) as compared to
the particles obtained by unmodified chitosan polymer which were
50-80 nm and non-spherical (FIG. 6E). The reduction in size is
because of the increase in amine groups due to the peptides, which
interacts more efficiently with negatively charged siRNA and thus
the amount of siRNA complexed increases as well. The increased
encapsulation efficiency yields smaller particles.
EXAMPLE II
In Vitro Transfection with the Nanoparticles
[0126] Transfection efficiency of the nanoparticles complexing
siGLO red (Cy3-labelled transfection indicator) was performed on
mouse neuroblastoma cells (Neuro 2a). 100% transfection efficiency
was achieved with chitosan-PEG-TAT nanoparticles encapsulating
siGLO-red in mouse neuroblastoma cells seeded at a density of 20
000 cells per well in a 96 well plate, supplemented with Eagles
minimum essential medium (EMEM) with 10% FBS (FIG. 7). The
fluorescence was achieved after 4 hours of transfection and was
observed under fluorescence microscope (Nikon Eclipse TE2000-U) at
a wavelength of 660 nm.
[0127] The cytotoxicity assay was performed using MTS assay on
cells seeded in 96 well plate after 4 hours of transfection with
different treatment groups (FIG. 8). The results indicate minimal
toxicity with chitosan nanoparticles modified with PEG alone and
with chitosan nanoparticles modified with PEG and peptide both as
compared to the unmodified chitosan nanoparticles. Without wishing
to be bound to theory, it is believed that the presence of PEG and
peptide which are hydrophilic in nature and pose no adverse or
toxic effect on the cells. It is also believed that PEG reduces the
steric-hindrance and TAT is an excellent cell-penetrating peptide.
It is also noted that chitosan polymer itself was observed to be
toxic to the cells, whereas anionic agent siGLO and the cross
linker TPP showed no/minimal adverse effects on the cells.
[0128] Plasmid purification: Ataxin-1 cDNA. The plasmid DNA
containing ataxin-1 cDNA, purified and resuspended in Tris-EDTA
buffered solution [pGFP-ataxin (Q82)] was a kind gift from Dr.
Zoghbi Huda, which was used for the transformation experiment in
DH5-alpha competent cells by means of chemical transformation also
known as heat shock transformation method. The procedure for
chemical transformation was followed according to the
manufacturer's protocol (Invitrogen). The colony selective
antibiotic for pGFP-ataxin was kanamycin (30 .mu.g/ml). Stock
cultures of transformed plasmid in DH5-alpha cells were made in
sterilized 80% glycerol and were stored at -80.degree. C. Plasmid
purification was performed following the protocol of Qiagen's maxi
kit. The purified plasmid obtained was quantified for its
concentration and purity. The percentage purity of the plasmid was
quantified using UV spectrophotometer. The ratio of Abs 260/280
obtained was 1.8 which signifies that the plasmid was purified and
the concentration obtained was 266 .mu.g/ml for pEGFP.ataxin1.
[0129] Overexpression/Suppression of ataxic proteins in Neuro2A
cells. Transfection studies were performed using a functional siRNA
against ataxin gene. 100 000 cells were seeded in a 12-well plate,
supplemented with EMEM containing 10% FBS. The cells were incubated
for 24 hours at 37.degree. C., 5% CO.sub.2. After 24 hours, the
media was replaced with FBS free EMEM medium and the cells were
transfected with pEGFP-ataxin1 at a concentration of 250 ng/.mu.l
using Lipofectamine 2000.TM. (Invitrogen) according to
manufacturer's protocol. Optimal transfection efficiency with
lipofectamine was determined experimentally. After 6 hours of
transfection, the media was replaced with complete growth EMEM
medium containing 10% FBS. Ataxin1-siRNA (Santacruz Biotechnology)
was complexed with chitosan-PEG-peptide nanoparticles as previously
described. The Ataxin1-siRNA transfection with nanoparticles was
performed on cells over-expressing ataxin protein. After 24 hours
of overexpression, 40 .mu.M of Ataxin1-siRNA complexed with
nanoparticles was used to transfect neuronal cells per well (12
well plate) containing FBS free EMEM medium. Further to
nanoparticles transfection after 6 hours, the media was again
replaced with complete growth EMEM medium containing 10% FBS. Empty
nanoparticles and nanoparticles containing scrambled siRNA (siGLO)
were used as positive controls. The cells were harvested for ataxin
protein estimation after 24 and 48 hours.
[0130] Protein extraction and Western Blot. To estimate the amount
of silencing by functional siRNA delivered through
Chitosan-PEG-peptide nanoparticles, cultured cells were washed
twice with cold PBS and lysed using 2.times. Laemmli Buffer (4%
SDS, 20% glycerol, 10% 2-.beta.-mercaptoethanol, 0.004% bromophenol
blue, 0.125 M Tris HCl, pH 6.8) at 4.degree. C. on an orbital
shaker. The cells were scrapped after 20 minutes (on ice). The
extracted protein samples were heated at 70 to 100.degree. C. for
10 minutes, before loading on gels. Procedure of western blot was
performed according to the manufacturer's protocol (Invitrogen).
Pre-cast Nupage Bis-Tris (4-12%) gels were used for running the
protein samples. Magic mark 1 Kb protein ladder was used as a
standard. The gel was elecrophoretically transferred to
nitrocellulose membrane using SemiDry blot apparatus
(Invitrogen).
[0131] The membrane was incubated for 1 hr in 5% non-fat powdered
milk in 0.2% TBST. The membrane was then incubated overnight with
goat ataxin-1 IgG polyclonal antibody (1:5 000 dilution; Santacruz
Biotechnology). After three washes in TBST, the membrane was
incubated with horseradish peroxidase (HRP)-conjugated donkey
anti-goat IgG antibody (1:2 000 dilution; Santacruz Biotechnology).
The membranes were then washed three times in TBST followed by
detection of signal with a chemiluminiscence detection kit (Roche)
(FIG. 9).
[0132] To control the protein loading, the membrane was reprobed
with antibody .alpha.-Actin (1:10 000; Santa Cruz Biotechnology).
As observed in FIG. 9, samples were run after 24 and 48 hours of
transfection. Sample A and C were positive controls with empty and
scrambled siRNA (siGLO) containing CS-PEG-TAT nanoparticles
respectively and sample B had Ataxin1-siRNA. The silencing was
observed after 48 hours in sample B. These results indicate that
nanoparticles prepared using the proposed synthetic scheme
successfully and efficiently delivered siRNA, leading to gene
silencing against ataxin 1 in neuronal cells in vitro.
EXAMPLE III
In Vivo Use of the Nanoparticles for Brain Delivery
[0133] Four weeks old C57BL/6J male mice weighing 10 to 15 g were
purchased from Jackson laboratories (Bar Harbor, Me., USA) and
housed in an environment with controlled temperature (22.degree.
C.), humidity, and a 12 h light/dark cycle at McGill's Animal care
facility. The animal experiment was conducted as per the protocol
approved by the Animal care committee at McGill University
(Montreal, QC, Canada). Standard mouse chow pellets and water were
supplied ad libitum. Animals were acclimatized for a week before
the start of the experiment.
[0134] Animal Study, tissue processing and staining. Animals were
randomized into 4 groups to receive treatment formulation
consisting of biotin tagged scrambled siRNA, complexed with
CS-PEG-peptide (TAT/MGF) nanoparticles (two animals/group). One
animal in each group received PBS and was treated as control.
CS-PEG-peptide (TAT/MGF) nanoparticle formulations complexing
biotin-siRNA were concentrated to 4 different doses at: (a) 0.25
mg/kg, (b) 0.5 mg/kg, (c) 1 mg/kg and (d) 2 mg/kg siRNA using
Amicon Ultra-15 centrifugal filters (MW cut-off 3 000 Daltons,
Millipore) prior to dosing. The animals were anesthetized with
75-100 .mu.l of cocktail comprising ketamine (100 mg/kg), xylazine
(10 mg/kg) and acepromazine (3 mg/kg) via intraperitoneal
administration. A total of 30 .mu.l of the nanoparticle formulation
was administered intranasally (2 .mu.l/drop) over 15-20 minutes
period, once a day. The experimental end points were 4, 16, 24 and
48 hrs. Before each end point the animals were anesthetized using
the above mentioned anesthetic cocktail and perfusion fixed with 4%
paraformaldehyde (PFA) (Sigma Aldrich, Canada). Brain, Lungs,
Heart, Stomach, Kidney and Liver were harvested and kept at
4.degree. C. in 4% PFA for 48 hrs. After which the tissues were
trimmed to 3 mm thick sections and stored at 70% ethanol in
histology cassettes. The tissues were paraffin embedded and
processed into 4 .mu.m thick section on slides at the histology
core facility (The Rosalind and Morris Goodman Cancer Research
Centre, McGill University). The tissue section on slides were
stained with Vectastain elite ABC kit (Vector laboratories;
Burlingame, Calif., USA) as per the manufacturer's protocol and
Diaminobenzidine (DAB) was used as a substrate to assess the
presence of biotin (brown staining). Hematoxylin was used as a
counterstain and slides were mounted with permount (Vector
laboratories; Burlingame, Calif., USA) and observed under compound
microscope (Leica DM500; Ontario, Canada) at .times.400
magnification.
[0135] FIG. 10(I) represents histopathological sections of brain
tissue (cerebral cortex) at 4 hrs time point with different doses,
containing (A) 0.25 mg/kg, (B) 0.5 mg/kg, (C) 1 mg/kg, (D) 2 mg/kg
and (E) PBS (control). The dark brown stained pyramidal neuronal
cells obtained with 0.5 mg/kg of scrambled biotin-siRNA complexed
nanoparticles, ensured the delivery of siRNA in the neuronal cells
of cerebral cortex (p=0.0001) and in the Purkinje cells of the
cerebellum (p=0.0001) as compared to the untreated control. Other
animals that received 0.25 mg/kg of scrambled biotin-siRNA showed
faint staining in the neuronal cells of cerebral cortex (p=0.006),
this is justified by the fact that dose A (0.25 mg/kg) was half the
concentration of dose B (0.5 mg/kg). Whereas, animals that received
1 and 2 mg/kg of scrambled biotin-siRNA dose, did not show any
staining in the tissue. The reason was interpreted, as while
concentrating the nanoparticle formulation from a range of 10-20 ml
solution to 30 .mu.l before dosing, the process of concentrating
led to clumping and aggregation of the nanoparticles, which
resulted in increase in nanoparticles size and it could not
penetrate the neurons.. FIG. 10(II) represents the quantitative
analysis of the tissues using ImageJ, which calculates the mean
percentage area of the dark brown stained cells using image J
software. Thus, this study determined that, under these
experimental conditions, the optimal dose for the developed
nanoparticles formulation administered intranasally was dose B at
0.5 mg/kg. In addition, these results indicated that the
nanoparticle formulation can be used for the successful delivery of
the biotin-siRNA with high efficiency and high target
selectivity.
[0136] FIG. 11(I) represents histopathological sections of brain
tissue (cerebral cortex) of mouse having received dose B (as
described in FIG. 10) at 0.5 mg/kg at different time points, (A) 4
hrs, (B) 16 hrs, (C) 24 hrs and (D) 48 hrs. As observed in the
figure, the staining with dose 0.5 mg/kg shows significant dark
brown staining in the pyramidal neurons of the cerebral cortex and
Purkinje cells of the cerebellum (p=0.0001) at 4 hrs (A). The
staining was observed only until 16 h in the cerebral cortex
(p=0.0001) and faded thereof, with no staining observed at 24 and
48 h. The result was quantified using Image J as represented in
FIG. 11(II). The time dependent study was conducted to determine
and monitor the expression of siRNA after being delivered by
nanoparticle formulation via intranasal route of administration.
These results indicate that, in these experimental conditions, the
nanoparticle formulation containing 0.5 mg/kg siRNA was
successfully delivered to neuronal cells within 4 hours. These
results also indicate that the intranasally administered
nanoparticle formulation is, under these experimental conditions,
cleared after 16 hrs.
[0137] FIG. 12A represents histopathological sections of different
organs with treatment dose B (as described in FIG. 10) comprising
nanoparticle formulation at siRNA dose 0.5 mg/kg (left column) and
control group with PBS (right column) after 4 hours. As observed in
the figure, the staining in the brain tissue was highly significant
with 0.5 mg/kg scrambled biotin-siRNA/nanoparticle dose in both
cerebral cortex and cerebellum (p=0.0001) and also with 0.25 mg/kg
but only in the cerebral cortex (p=0.006), as represented in FIG.
4B. The staining with biotin-siRNA/nanoparticle dose at 0.5 mg/kg
was also observed to target heart sarcomeres (p<0.01), with
significance as compared to other dose concentrations; 0.25 mg/kg
(p=0.403), 1 mg/kg (p=0.562), 2 mg/kg (p=0.999) (FIG. 12B). Renal
cells in the medulla region of the kidney and hepatic cells also
showed brown-colored staining in the cells, with 0.5 mg/kg of
scrambled biotin-siRNA/nanoparticle formulation (p=0.0001) as
compared to the untreated control (FIG. 4B). The glandular cells of
the stomach and alveoli in lungs showed no significant difference
as compared with the untreated control. Among all the
concentrations of different treatment doses tested, the highest
staining was observed with 0.5 mg/kg of scrambled
biotin-siRNA/nanoparticle formulation in the cerebral cortex and
cerebellum (p<0.01), when compared with staining in other
organs, except the heart. FIG. 13 represents the enlarged view of
cerebral cortex and purkinje cells. However, in the cerebral
cortex, the anterior olfactory bulb, hippocampus, thalamus,
hypothalamus were also found to be well stained.
[0138] As per the nanoparticle formulation of CS-PEG-TAT/MGF, the
MGF peptide was used as targeting ligand for purkinje cells. The
MGF peptide is a splice variant of IGF and has been shown to have
affinity towards neuronal cells and myocardial tissues. Thus the
staining in the heart could be the due to the affinity of MGF
peptide targeting the heart sarcomeres. Following the intranasal
route of administration, a fair amount of nanoparticles was
expected to target lungs through the airway pathway. Renal, hepatic
cells have been shown to have stained as compared to the control
sections. It is to be noted that no staining was observed in any
tissue sections at 24 hrs and 48 hrs time point. This could be due
to the degradation of the biotin-siRNA molecule by that time and at
16 h, except brain tissues (cerebral cortex) no other tissue
section was observed to have staining in the cells.
[0139] Cytotoxicity--TUNEL assay on animal tissues. Analysis of
apoptotic cells in tissues was performed using Terminal
deoxynucleotidyl transferase-mediated dUTP nick-end labeling
(TUNEL) staining (Promega, Madison, Wis., USA) as per the
manufacturer's protocol after a pretreatment procedure performed to
block biotin-siRNA. In brief, the tissue sections in paraffin block
were dewaxed in xylene and rehydrated in decreasing concentrations
of ethanol and washed with PBS. The tissue sections were then
incubated with Streptavidin-HRP reagent (Roche Diagnostics) for 10
minutes, washed in PBS. After which the sections were incubated
with 3% H.sub.2O.sub.2 for another 10 minutes and again washed in
PBS. This step was included to block biotin-siRNA already present
in the neuronal cells and to avoid any false positive results.
After this step, the protocol of TUNEL assay was performed as per
the manufacturer's instructions. The tissue sections were
counterstained with hematoxylin, washed in distilled water,
dehydrated and mounted with permount (Vector laboratories;
Burlingame, Calif., USA) to be observed under compound microscope
(Leica DM500; Ontario, Canada) at .times.400 magnification.
[0140] FIG. 14 represents histopathological sections of different
organ tissues from animal receiving nanoparticle formulation
containing biotin tagged scrambled siRNA as a dose at 0.5 mg/kg of
animal weight, euthanized after 4 hrs. The tissues did not show any
apparent staining in the cells, which confirms no
toxicity/apoptosis of cells due to the administration or presence
of the nanoparticle formulation.
[0141] Without wishing to be bound to theory, FIG. 15 represents a
potential mechanism of cellular uptake of surface functionalized,
nanoparticle through a targeted receptor-mediated endocytosis
pathway, with endocytotic release from the endosome due to "proton
sponge" effect. Following the endosomal disruption, the delivered
therapeutic molecule gets released intra-cellularly.
EXAMPLE IV
In Vivo Use of the Nanoparticles for the Treatment of Alzheimer's
Disease
[0142] Neurodegenerative diseases are characterized by progressive,
age-related loss of specific subsets of neural cells, which lead to
diverse clinical phenotypes depending on the underlying anatomical
involvement. The etiology of neurodegenerative diseases is most
often multifactorial, likely a result of gene-environmental
interaction, which may lead diseases like Parkinson's, Alzheimer's,
Huntington's, Amyotrophic lateral sclerosis (ALS) and
Spinocerebellar Ataxia (SCA). These diseases tend to progress
slowly over the time and generally target older population.
Alzheimer's is the most common form of dementia, which is incurable
and degenerative. It is predicted to affect 1 in 85 people globally
by 2050. As per the 2008 clinical trial report, it is uncertain if
any of the intervention strategies tested among 500 clinical
trials, are likely to show promise for the identification and
treatment of Alzheimer's disease (AD). The mean life expectancy of
an individual is drastically reduced to 7 years after the onset of
the disease. Currently, there is no cure available that can halt
the progression of the disease. However, therapies and drugs are
available in the markets that merely mitigate the symptoms of the
disease.
[0143] Alzheimer's disease is characterized by the presence of
misfolded protein in the form of senile plaques and neurofibrillary
tangles in the brain. The senile plaques are composed of dense,
insoluble deposits of amyloid-beta peptide in and around the
neuron. These peptides are the fragment of a larger transmembrane
protein called amyloid precursor protein (APP). APP is essential
for neurons growth survival and post injury repair. The formation
of amyloid-beta peptides is initiated by a sequential cleavage of
APP by an enzyme, protease beta-secretase, also known as BACE1
(beta site APP cleaving enzyme) and then by a gamma-secretase, an
aspartyl protease complex, which generates toxic C-terminal
fragments inside the cell and releases a fragment called
amyloid-beta peptide extra cellularly. Neurofibrillary tangles are
aggregates of the microtubule-associated tau protein which has
become hyperphosphorylated and accumulate inside the cells. The
underlying pathological mechanism of AD is still unknown but the
accumulation of amyloid-beta peptides is thought to be the central
triggering event of the disease, which is believed to disrupt the
cell's calcium ion homeostatis, leading to apoptosis. Amyloid-beta
is also known to accumulate in the mitochondria of affected cells
and inhibit certain enzyme functions that further inhibit the
utilization of glucose by neurons. Moreover, alteration in the
distribution and expression of brain derived neurotrophic factors
(BDNF) has also been observed to be associated with AD.
[0144] The identification of these biological and pathological
abnormalities triggered the studies on recognizing the genes
responsible for causing inherited forms of AD. On such form is
autosomal dominant familial Alzheimer's disease, which is caused
due to the mutations in the APP and components of gamma-secretase
(Presenilins 1 and 2) that lead to increased production of
amyloid-beta 2 protein, the main component of senile plaques.
Several transgenic animal models have been developed based on
various genetic mutations to understand the aetiology and possible
pathological mechanism of the disease and to investigate various
therapeutic options.
[0145] Advancement in RNAi therapy has facilitated the
understanding of pathobiological mechanisms of the disease with
most of the researches focusing on phenotype rescue due to
dominantly acting mutant genes and loss-of function analysis. Based
on the aetiology of the AD, the key targets for RNAi therapy are
assumed to be APP, BACE and gamma-secretase and tau, which can
eliminate the production of toxic C-terminal fragments and
amyloid-beta peptides. siRNAs has been delivered to the central
nervous system both naked or with the help of some transfection
reagents in-vivo, targeting different molecular targets in
different parts of the nervous system, showing effective gene
silencing. Direct doses of siRNAs administered intrathecally or
intracerebro-ventricularly pose a widespread inhibition of
molecular targets that are broadly expressed in different parts of
the brain but may also lead to off-targeting. A significant
limitation is the inability of the therapeutic molecules to cross
the blood-brain barrier and other physiological barriers. Thus the
application of a novel therapeutic modality also depends on the
development of an efficient and clinically feasible means of
administration. Though, various siRNA delivery strategies have been
explored, but they have not proved to be as effective and have
created concerns with safety issues, thus a polymeric approach of
delivering siRNA molecules, which is target specific,
multifunctional, biodegradable and biocompatible is more appealing.
We have developed a self-assembled, functionalized receptor
targeting nanoparticles from chitosan that are biodegradable and
biocompatible in nature and perform target specific delivery of
siRNA, when delivered intranasally to the cerebral cortex of the
brain.
[0146] Materials. Chitosan (M.W. 7 to 10 KDa) with a viscosity of 5
to 20 cP (at room temperature) and a degree of deacetylation of
80.0% was purchased from Wako (Richmond, Va., USA). Polyethylene
glycol monomethyl ether (mPEG): medium molecular weight (2 000 Da),
phthalic anhydride, pyridine, toluene, hydrazine monohydrate,
succinic anhydride, ethanethiol, aluminium chloride, sodium
tripolyphosphate (TPP), and glacial acetic acid of analytical grade
are obtained from Sigma (Oakville, ON, Canada). Anhydrous
N,N-Dimethylformamide (DMF),
1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride) (EDC),
4-(Dimethylamino) pyridine (DMAP), sodium hydroxide (NaOH),
methanol, diethyl ether, chloroform, concentrated hydrochloric acid
were obtained from Thermo Fisher Scientific (Ottawa, ON, Canada).
Thionyl chloride (SOCl.sub.2) is obtained from VWR (Mississauga,
ON, Canada). TAT peptide (NH.sub.2-RKKRRQRRR-NH.sub.2) (SEQ ID NO:
4) M.W. 1339.63 and MGF peptide
(NH.sub.2-YQPPSTNKNTKSQRRKGSTFEEHK-NH.sub.2) (SEQ ID NO: 1) M.W.
2848.14, were synthesized by Sheldon Biotech, McGill University.
siRNA targeting PSN1 (Accession #: NM_000021.3) targeting sequence:
5' AAG GUC CAC UUC GUA UGC UGG (SEQ ID NO: 5) was synthesized from
Dharmacon (Lafayette, Colo., USA).
[0147] Synthesis of peptide tagged PEGylated chitosan polymer and
nanoparticles preparation. The polymer CS-PEG-TAT/MGF was
synthesized as described in Example I. The nanoparticles were
prepared from the chemically modified polymer (CS-PEG-TAT/MGF). The
formation of nanoparticles involved ionic-gelation method, as
previously described by Calvo et al. (1997). The optimization of
nanoparticles on the basis of size and surface charge has been
previously performed and is published elsewhere (Malhotra et al.,
1999). In brief, 0.5 mg/ml of chemically modified polymer was
crosslinked with 0.7 mg/ml of sodium tripolyphosphate (TPP),
complexing 2 .mu.g/ml of siRNA.
[0148] Animal model. Four weeks old Alzheimer's transgenic mice
model Tg(APPSwFILon,PSEN1*M146L*L286V)6799Vas were purchased from
MMRC facility in Jackson Laboratory (Bar Harbor, Me., USA). These
transgenic mice over-express both mutant human APP(695) with the
Swedish (K670N, M671 L), Florida (1716V), and London (V7171)
Familial Alzheimer's Disease (FAD) mutations and human PS1
harboring two FAD mutations, M146L and L286V. This transgenic mice
model was chosen as it over expresses amyloid-beta 42 protein in
cerebral cortex and hippocampus, resulting in amyloid plaque
pathology as early as 2 months of age. Other characteristic
features include; reduced synaptic markers, increased p25 levels
and neuron loss.
[0149] The animals were housed in an environment with controlled
temperature (22.degree. C.), humidity, and a 12 h light/dark cycle
at McGill's Animal care facility. The animal experiment was
conducted as per the protocol approved by the Animal care committee
at McGill University (Montreal, QC, Canada). Standard mouse chow
pellets and water were supplied ad libitum. Animals were
acclimatized for two weeks before the start of the experiment. The
animals were divided in two groups. The first group (n=4) received
siRNA against PSN1 gene and the control group (n=4) received no
treatment at all. The treatment began at 6 weeks of animal age. The
siRNA dose administered through nanoparticles was 0.5 mg/kg of
animal weight. Prior to the dose administration the animals were
anesthetized with isoflurane, gas anesthesia, as per the animal use
protocol of McGill University. The treatment dose was administered
daily, intranasally, for a total of 3 weeks. All the animals were
sacrificed at the end of 3 weeks. Brain was harvested for PSN1 mRNA
quantification.
[0150] Percentage mRNA knockdown by siRNA delivered through the
nanoparticles. To further validate the knockdown results of the
endogenous PS1 expression in the treatment group, when compared to
the control, a quantitative real time PCR was performed. Briefly,
the brain tissue excised at the end point was preserved in
RNAlater.RTM. RNA stabilization reagent from Qiagen and stored at
-20.degree. C., until use. The total RNA was extracted using
RNAeasy.RTM. Lipid tissue mini kit from Qiagen and the total RNA
will be quantified using Nanodrop 2000 spectrophotometer. The
reverse transcription on total RNA was performed to obtain cDNA
using a QuantiTect.TM. Reverse Transcription kit from Qiagen. A
quantitative real time PCR was performed using MBI Evolution
Evagreen.TM. Master Mix following the manufacturer's protocol (MBI,
Montreal, Canada) on ECO RT PCR machine from Illumina. The relative
expression levels of the targeted gene were compared with the
housekeeping gene GAPDH. The primer sequences used were as follows:
PSN1: F 5'-CCGAAATCACAGCCAAGA-3'; (SEQ ID NO: 6) R:
5'-CATTCACAGAAGATACCAAGAC-3'. (SEQ ID NO: 7) GAPDH, F:
5'-TAAAGGGCATCCTGGGCTACACT-3'; (SEQ ID NO: 8) R:
5'-TTACTCCTTGGAGGCCATGTAGG-3' (SEQ ID NO: 9). The PCR was run for
40 cycles with a 95.degree. C. denaturing step (15 s), a 56.degree.
C. annealing step (1 min), and a 72.degree. C. extension step (15
s), plus final incubation at 72.degree. C. for 10 min.
[0151] Evaluation of percentage gene knockdown of PSN1 gene,
silenced by siRNA delivered intranasally via novel
receptor-targeted nanoparticles. The PSN1 gene knockdown was
evaluated by extracting total RNA form the brain tissues, then
reverse transcribing it to form a cDNA and then running a Real-Time
PCR on cDNA using primers specific for PSN1 gene. The percentage
gene knockdown of PSN1 gene was evaluated by comparing the
treatment group with the untreated control. As represented in FIG.
16, a 21.34% of gene knockdown was achieved in the treatment group
(n=4) that received siRNA against PSN1 gene, complexed with
nanoparticles, as compared to the untreated control (n=4). Though
not statistically significant (p=0.162), these results propose a
promising approach of delivering siRNA using receptor-targeted
nanoparticles via intra-nasal route for brain targeting.
[0152] Systemic safety and toxicity of novel siRNA-nanoparticle
formulation. Safety markers were analyzed from the serum collected
from the animals that received 0.5 mg/kg of PSN1-siRNA, complexed
with peptide tagged PEGylated chitosan nanoparticles. The liver
function and toxicity tests were performed using alkaline
phosphatase (ALP) and aspartate aminotransferase/alkaline
aminotransferase (AST/ALT) respectively. The results for ALP though
show a significant difference between the treatment and control
group (FIG. 17, p=0.036) but had values that fall in the normal
range of 44 to 147 IU/L. For AST/ALT, the results indicate no
significant difference between the treatment and the control group
(FIG. 17, p=0.379). Another test performed was CRP, which is an
indicative of systemic inflammation the results showed no
significant difference between the treatment and the control (FIG.
17, p=0.472). Urea, CRE and UA tests were performed indicating the
renal function in animals. The results indicate no significant
difference in urea (FIG. 17, p=0.535) and UA (FIG. 17, p=0.737)
levels, however the creatinine was found to be elevated in the
treatment group as compared to the control (FIG. 17, p=0.025), but
the values in both the groups were all found to be in the normal
range i.e. 38.13 to 91.5 .mu.mol/L.
EXAMPLE V
In Vivo Use for Treating Cancer
[0153] Cancer is characterized by uncontrolled growth of group of
cells that infest adjacent tissues and often metastasize to other
organs via lymphatic system or blood stream. Cancer is primarily
caused by environmental factors (90-95%) and few with genetic
(5-10%). The uncontrolled growth of group of cells in the case of
cancer is usually triggered by malfunctioning of the genes that
manipulate cell's growth and differentiation. Typically the
alteration in cell growth promoting, oncogenes and cell division
inhibiting, tumor suppressive genes lead to the formation of cancer
cells. The genetic causes of cancer are usually due to gain or loss
of an entire chromosome due to errors in mitosis or changes in
nucleotide leading to mutations in the genomic DNA. Depending on
the stage of the cancer the treatment options available include
surgical removal, chemotherapy with anticancer drugs, such as
5-fluorouracil, oxaliplatin and leucovorin, radiation therapy,
immunotherapy and hormone block therapy with drugs like cetuximab
and panitumumab. However, it has been shown that cancers with
genetic origin are not benefited with these chemotherapies.
Moreover, the toxicity and side effects has severely limited the
safety and effectiveness of these methods. One of the target
proteins in cancer therapy is PLK1, which is a serine/threonine
kinase, a key regulator for mitosis in mammalian cells. It is
required for centrosome maturation, bipolar spindle formation, and
chromosomal segregation and is also associated with microtubules
and centrosomes at various stages of mitosis. PLK1 is considered as
a proto-oncogene, which over-expresses in a variety of human
cancers. PLK1 is directly associated with p53, a tumor suppressor
protein. PLK1 on interaction with p53 inhibits its transactivation
activity and pro-apoptotic activity, leading to uncontrolled
proliferation of cells. Lately, inhibition of PLK1 with antibodies,
antisense oligonucleotides (ASO's), small interfering RNA (siRNA)
or dominant negative mutants that suppress the tumor growth by
causing increased apoptosis has gained much interest as a
therapeutic option to treat tumor diseases. Although,
antineoplastic drugs have shown great success as a treatment for
cancer therapy, many carcinomas are resistant to these agents and
thus, chemotherapy with these agents has become a major restriction
at an advanced cancer stage. Therefore, siRNAs targeted against
proliferation-associated signal transduction pathways, which can
halt the tumor progression in animal models is emerging as an
appealing approach. For cancer therapy, a receptor-targeted
nanoparticles for in vivo delivery of siRNA has been developed. The
target specificity of the nanoparticles is attributed to a peptide
that guides the nanoparticle system carrying siRNA to specific
tissue, when administered via systemic route. The advantage of
using peptide based tumor targeting is their rapid clearance from
the blood because of their small size and lack of immunogenicity.
The identification of peptides for tumor-specific targeting has
been facilitated by a technology called, phage display library,
which takes into account the ability of filamentous bacteriophage
to present large number of peptides and proteins on their surface,
allowing these specific peptide sequences to bind to the target
specific tumors or cell types. This method for identification of
specific binding ligands has found wide application in isolating
peptides that has high binding affinity towards cancer cells. The
conjugation of these peptides to conventional chemotherapeutic
drugs, diagnostic/imaging molecules and nanoparticles will enable
their delivery in low dose with effective targeting. In this study,
the peptide CP15 with a sequence NH.sub.2-VHLGYAT-NH.sub.2, which
was identified by the technology of phage displayed library was
used. CP15 peptide has shown to be the most effective peptide
targeting colon tumor cells but not the normal human intestinal
epithelial human cells. The novel receptor-targeted nanoparticle
formulation developed in this study were tagged with CP15 peptide
as a ligand that will guide the nanoparticles to selectively target
the tumor tissue expressing receptors for CP15 peptide in a mouse
xenograft model of colon cancer developed from SW480 epithelial
colon cancer cells. This illustrates the potential of using these
novel receptor-targeted nanoparticles to be used in future for
cancer therapy.
[0154] Materials. Chitosan (M.W. 50 KDa to 190 KDa) with viscosity
20 to 300 cP (at room temperature) and degree of deacetylation of
75 to 85% was obtained from Sigma (Oakville, ON, Canada).
Polyethylene glycol monomethyl ether (mPEG): medium molecular
weight (2 000 Da), phthalic anhydride, pyridine, toluene, hydrazine
monohydrate, succinic anhydride, ethanethiol, aluminium chloride,
sodium tripolyphosphate (TPP), agarose, ethidium bromide (10 mg/ml)
and glacial acetic acid of analytical grade are obtained from Sigma
(Oakville, ON, Canada). Anhydrous N,N-Dimethylformamide (DMF),
1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride) (EDC),
4-(Dimethylamino) pyridine (DMAP), sodium hydroxide (NaOH),
methanol, diethyl ether, chloroform, concentrated hydrochloric acid
were obtained from Thermo Fisher Scientific (Ottawa, ON, Canada).
Thionyl chloride (SOCl.sub.2) is obtained from VWR (Mississauga,
ON, Canada). CP15 (NH.sub.2-VHLGYAT-NH.sub.2) M.W. 758.3, were
synthesized by Sheldon Biotech, McGill University. Pre-cast Nupage
4 to 12% Bis-Tris gels, Nupage MES running buffer, Nupage transfer
buffer, Nupage LDS sample buffer (4.times.), nitrocellulose
membrane 0.45 .mu.m, blotting filter paper and Magic Mark.TM. (1
Kb) protein ladder were obtained from Invitrogen (Burlington, ON,
Canada). Scrambled/non-targeting (NT) siRNA tagged with biotin, and
PLK siRNA (h) with sequence; PLK (sense strand)--5'
AGAmUCACCCmUCCUmUAAAmUAUU 3' (SEQ ID NO: 10) and PLK (antisense
strand)--5' UAUUUAAmGGAGGGUGAmUCUUU 3' (SEQ ID NO: 11) from
Dharmacon (Lafayette, Colo., USA), where "m" represents
2'Omethylated nucleotide. Primary antibodies; PLK1 (F-8) mouse
monoclonal antibody, .beta.-Actin (C-4) mouse monoclonal antibody,
probed with secondary antibodies; (HRP)-conjugated goat antimouse
antibody. All the antibodies were procured from Santacruz
Biotechnology (Santa Cruz, Calif., USA).
[0155] Synthesis of peptide (CP-15) tagged PEGylated chitosan
polymer. The polymer CS-PEG-CP15 was synthesized as described in
Example I. The modified polymer was characterized and analyzed
using .sup.1H (Mercury 400 and 500 MHz NMR) and Transmission
Electron Microscopy (Philips EM410 TEM).
[0156] Preparation of novel polymeric nanoparticles from CP-15
tagged PEGylated chitosan polymer. Nanoparticles were prepared from
the chemically modified polymer (CS-PEG-CP-15). The formation of
nanoparticles involved ionic-gelation method, as previously
described by Calvo et al. (1997). The optimization of nanoparticles
on the basis of size and surface charge has been previously
performed and is published elsewhere (Malhotra et al., 2009). In
brief, 0.5 mg/ml of chemically modified polymer was crosslinked
with 0.7 mg/ml of sodium tripolyphosphate (TPP), complexing 8
.mu.g/ml of siRNA. The characterization of nanoparticles size and
dispersivity was analyzed by transmission electron microscopy (TEM)
and the siRNA loading efficiency was determined by gel retardation
assay.
[0157] Animal study and in vivo tumor induction. Six weeks old mice
Balb/c nude mice, weighing 15 to 20 g were purchased from Charles
River Laboratories (Wilmington, Mass., USA) and housed in an
environment with controlled temperature (22.degree. C.), humidity,
and a 12 h light/dark cycle at McGill's Animal care facility. The
animal experiment was conducted as per the protocol approved by the
Animal care committee at McGill University (Montreal, QC, Canada).
Standard mouse chow pellets and water were supplied ad libitum.
Animals were acclimatized for a week before the start of the
experiment. For tumor induction, animals were subcutaneously
inoculated with 100 .mu.l of SW480 colon cancer cells
(2.times.10.sup.6) mixed with an equal volume of Matrigel.TM. (BD).
The treatment began after the tumor reached a volume of 100
mm.sup.3. The animals were randomized into 4 treatment groups (n=7)
to receive treatment formulations. The following treatment
formulations: 1) CS-PEG-CP15 with PLK1 siRNA; 2) CS-PEG-CP15 with
non-targeting (NT) siRNA, 3) PLK1 siRNA alone and 4) Untreated
control. In each treatment group the animals received a total siRNA
dose of 0.5 mg/kg. 100 .mu.l of treatment formulations were
administered thrice a week via intra-peritoneal injections for a
period of 2 weeks.
[0158] Histopathological analysis to identify siRNA delivered via
novel nanoparticles in tumor tissue. One animal from each treatment
group was euthanized by CO.sub.2 asphyxiation, after 4 hours of
first dose administered via intra-peritoneal route. Brain, Lungs,
Heart, Kidney, Spleen and Liver were harvested and kept at
4.degree. C. in 10% phosphate buffered formalin for 48 h. After
which the tissues were trimmed to 3 mm thick sections and stored at
70% ethanol in histology cassettes. The tissues were paraffin
embedded and processed into 4 .mu.m thick section on slides at the
histology core facility (The Rosalind and Morris Goodman Cancer
Research Centre, McGill University). The tissue section on slides
were stained with Vectastain Elite ABC.TM. kit (Vector
laboratories; Burlingame, Calif., USA) as per the manufacturer's
protocol and Diaminobenzidine (DAB) was used as a substrate to
assess the presence of biotin, which was used as a tag on siRNA for
histology identification purposes. Hematoxylin was used as a
counterstain and slides were mounted with Permount.TM. (Vector
laboratories; Burlingame, Calif., USA) and observed under compound
microscope (Leica DM500; Ontario, Canada) at .times.400
magnification.
[0159] RNA extraction and QPCR to determine percentage gene
knockdown by siRNA targeted against PLK1 gene delivered through
novel nanoparticles. To further validate the knockdown effects of
endogenous PLK1 expression after nanoparticles based siRNA delivery
against PLK, a quantitative real time PCR was performed. Briefly,
the tumor tissues excised at the end point were preserved in
RNAlater.RTM. RNA stabilization reagent from Qiagen (Toronto, ON,
Canada) and stored at -20.degree. C. The total RNA was extracted
using RNeasy.RTM. Plus mini kit from Qiagen, following the
manufacturer's protocol. The total RNA was quantified using
Nanodrop 2000 spectrophotometer from Thermo Scientific (Rockford,
Ill., USA). Reverse transcription was performed to obtain cDNA from
1 .mu.g of total RNA using a QuantiTect.TM. Reverse Transcription
kit from Qiagen. Following that quantitative real time PCR was
performed using MBI Evolution Evagreen Master Mix.TM. following the
manufacturer's protocol (MBI, Montreal, Canada) on ECO RT PCR
machine from Illumine (San Diego, Calif., USA). The relative
expression levels of PLK1 gene were normalized with the
housekeeping gene GAPDH. The primer sequences used were as follows:
PLK1 Plk1, 5'-GGCAACCTTTTCCTGAATGA-3' (SEQ ID NO: 12) and
5'-AATGGACCACACATCCACCT-3' (SEQ ID NO: 13); GAPDH,
5'-TAAAGGGCATCCTGGGCTACACT-3' (SEQ ID NO: 14) and
5'-TTACTCCTTGGAGGCCATGTAGG-3' (SEQ ID NO: 15). The PCR was run for
30 to 40 cycles with a 95.degree. C. denaturing step (5 s), a
60.degree. C. annealing step (15 s), and a 72.degree. C. extension
step (15 s), plus final incubation at 72.degree. C. for 10 min.
[0160] Protein expression after siRNA delivered through novel
nanoparticles. After 2 weeks of treatment, the animals were
sacrificed as per the approved protocol by McGill University and
the tumor tissues were harvested and preserved at -20.degree. C. in
All Protect Tissue Reagent.TM. from Qiagen (Toronto, ON, Canada).
The tissue samples were sliced into small pieces and homogenized
using PowerGen Model 125 Homogenizer from Fisher Scientific
(Ottawa, Ontario, Canada) at 26 300 rpm in 2 mL of ice cold RIPA
buffer (20 mM Tris pH 8, 150 mM NaCl, 5 mM EDTA, 1% Nonidet P-40,
0.1% SDS, 10.0% Glycerol, mM Na.sub.2HPO.sub.4.7H.sub.20, 1% Sodium
deoxycholate) containing phenylmethylsulfonyl fluoride (PMSF) and
protease inhibitor cocktail from Roche Diagnostics (Laval, QC,
Canada). The crude extract was incubated on ice for 30 minutes and
centrifuged at 10 000.times.g for 10 minutes at 4.degree. C. to
remove tissue debris. The supernatant was collected and the protein
concentration was determined using Pierce.RTM. BCA Protein assay
kit from Thermo Scientific (Rockford, Ill., USA). Briefly, aliquots
containing 100 .mu.g of protein were heated at 70.degree. C. for 15
minutes with Nupage LDS sample buffer supplemented with 100 mM DTT.
The proteins were fractionated on precast NuPAGE.RTM. 4 to 12%
Bis-Tris Gel from Invitrogen (Ontario, Canada) at 200 V for 35
minutes in MES SDS running buffer. Magic Mark.TM. 1 Kb protein
ladder was used as a standard. The gel was electrophoretically
transferred to 0.45 .mu.m pore size Novex.RTM. nitrocellulose
membrane using Nupage transfer buffer on a Novex.RTM. SemiDry
blotter (Invitrogen, ON, Canada). After transfer the nitrocellulose
membrane was incubated for 1 hr in 5% non-fat powdered milk in
1.times. Tris buffered saline (TBS) buffer supplemented with 0.2%
Tween 20. The membrane was then incubated overnight at 4.degree. C.
with mouse PLK (F-8) monoclonal antibody (1:100 dilution). The next
day the membrane was washed thrice with TBST for 15 minutes each,
and then incubated with (HRP)-conjugated goat antimouse IgG
secondary antibody (1:2,000 dilution). The membrane was again
washed thrice with TBST followed by detection of signal with
chemiluminiscent agents (ECL, Amersham) from GE healthcare. The
bound antibody was visualized using autoluminography. To control
the protein loading, the membrane was reprobed with primary mouse
monoclonal .beta.-Actin (C-4) antibody (1:1 000) with an overnight
incubation at 4.degree. C., followed by subsequent three washes in
TBST and detection with HRP-conjugated goat anti-mouse IgG
secondary antibody (1:2 000) and development with chemiluminiscent
agents as described earlier. The protein band intensities were
quantified using Image J software.
[0161] Serum collection and analysis. Serum was collected through
jugular vein just before the end-point using a sterile 23 G/25 mm
needle and approximately 200 .mu.l of blood was collected in
Microtainer.RTM. serum separator tubes (Becton Dickinson, N.J.,
USA). The blood was allowed to clot at room temperature for 30
minutes and subsequently placed on ice until centrifugation. Serum
was separated by low-speed centrifugation at 3600 rpm for 8 min at
4.degree. C. The separated serum was frozen at -85.degree. C. until
analysis. Serum was used to test C-reactive protein (CRP) and liver
function tests such as alanine aminotransferase (ALT) and aspartate
transaminase (AST) using a conventional enzymatic method on Hitachi
911 automated clinical chemistry auto-analyzer (Roche Diagnostics,
USA).
[0162] Statistical analysis. Statistical analysis was carried out
using Graphpad Prism.TM. version 5.0 for windows (GraphPad
software, San Diego, Calif.). Values are expressed as
means.+-.Standard Error. The statistical comparison between
treatment groups was performed using ANOVA Tukey's test. P
values<0.05 were considered significant.
[0163] Synthesis of CP-15 tagged PEGylated chitosan polymer and
preparation of nanoparticles. The peptide-tagged PEGylated chitosan
polymer was synthesized following a series of chemical reactions as
represented in FIG. 1. Each intermediate step and the final product
of the synthesis were characterized for the functional group
modification and substitution by FTIR and .sup.1H NMR as optimized
in Malhotra et al. (2009). FIG. 18 illustrates the NMR spectra of
the final product obtained (CS-PEG-CP15) after the synthesis. The
multiple peaks of oxymethyl groups in PEG at .delta. 3.3 to 3.7
cover over the signals of pyranose ring of chitosan in the spectra.
The weak and broad peak at .delta. 4.3-4.5 are from the protons of
--NH--CH(CH.sub.2)--CO-- in CP-15 peptide. The multiple peaks at
.delta. 6.0-9.0 belong to the CP-15 peptide sequence.
[0164] Characterization of nanoparticles. Nanoparticles were
prepared following an ionic gelation scheme, wherein the cationic
polymer complexes the anionic molecule due to electrostatic
interaction. FIG. 19 represent nanoparticles as observed under TEM.
The nanoparticles obtained were polydispersed and ranged from 100
to 200 nm in size. The polydispersivity observed in nanoparticles
size depends on the average molecular weight of the polymer used.
Molecular weight corresponds to the chain length of the polymer. In
this example, chitosan of molecular weight 50 KDa to 190 KDa was
used as a parent polymer on which further chemical modifications
and substitution of a hydrophilic polymer, PEG and a cell
targeting/penetrating peptide (CP15) was performed. Since, each
monomer of a chitosan polymer comprises of an amine group, thus
larger chain length contributes to more number of amine groups. The
presence of amine groups adds a positive character to the polymer,
which enables it to complex a negatively charged molecule, such as
siRNA. Due to the increase in the polymer chain length of chitosan
as compared to the other low molecular weight chitosan (M.W. 10
KDa) used for brain study (Example I to III), it was anticipated
that the nanoparticle formulation will be able to complex more
siRNA. To confirm this assumption, a gel retardation assay was
performed (FIG. 20) and it was observed that the nanoparticle
formulation was able to complex 4 times higher amount of siRNA (8
.mu.g/ml) than the previous formulation (2 .mu.g/ml).
[0165] Evaluation of the ability of the receptor-targeted
nanoparticles to systemically deliver siRNA at the targeted tumor
site. Novel receptor-targeted nanoparticles were evaluated for
their ability to deliver siRNA at the targeted tumor site through
systemic route. The animals were sacrificed after 4 hours of first
dose administered intra-peritoneally. The treatment formulation
prepared from a chemically modified polymer, CS-PEG-CP15 was
complexed with scrambled biotinylated siRNA at 0.5 mg/kg and was
evaluated for its efficiency in comparison to siRNA delivered
without nanoparticles. FIG. 21 represents histopathological
staining from a mouse xenograft model of SW480 colon cancer. The
tumor tissue having the scrambled biotinylated siRNA stained dark
brown in color with Vecta.TM. stain elite kit, using DAB as a
substrate. The results shown on FIG. 21(I) indicate that the
staining was found to be approximately equal for both (a)
CS-PEG-CP15-siRNA nanoparticle formulation (p=0.00043) and (b)
unmodified chitosan-siRNA nanoparticle formulation (p=0.0011). The
expression of (c) scrambled biotin-siRNA, delivered alone was
comparatively less and was not significant, when compared to the
untreated control (p=0.062) FIG. 21(II) represents the mean
percentage area analysed for intensity in triplicates from an
animal tissue in each treatment, using Image J software.
[0166] FIG. 22A represents the biodistribution analysis of the
above mentioned 3 treatments in various other organs: heart, lungs,
kidney, liver and spleen and were compared with the untreated
control. The results indicate significant biodistribution of the
unmodified chitosan nanoparticles in all the organs (p<0.05)
when compared with the control. For CS-PEG-CP15 with siRNA
nanoparticle formulation, the biodistribution was found to be
significant only in heart (p=0.001) and lungs (p=0.017), as
compared with the control. Likewise, siRNA delivered alone also
showed significant expression in heart (p=0.009) and lungs
(p=0.043). FIG. 22(II) represents the mean percentage area analysed
for intensity in duplicates from an animal tissue in each
treatment, using Image J software.
[0167] The tumor accumulation data as represented in FIG. 21,
suggests that the derivatized chitosan nanoparticles were able to
target the tumor tissue as effectively as the unmodified chitosan
nanoparticles, when compared with the siRNA delivered alone and the
untreated control. This data suggests that the importance and
efficiency of chitosan polymer as a carrier to deliver the siRNA at
the targeted site. Thus, it can be inferred that the systemic tumor
targeting, could majorly be a size dependent phenomenon due to the
EPR effect rather than receptor mediated targeting. However, the
biodistribution study as presented in FIG. 22 showed that the
CS-PEG-CP15 nanoparticles accumulated less in other organs, when
compared to the unmodified chitosan nanoparticles (kidneys,
p<0.046). Thus, it can be inferred that the presence of a
targeting moiety on the nanoparticles restricted their uptake by
other tissues [41]. The enhanced circulation and lack of
accumulation in other organs of CS-PEG-CP15 nanoparticles is
attributed to the incorporation of PEG, which caters to the
increased stability of the nanoparticles in blood, without being
degraded or filtered by kidneys. These results indicate that these
nanoparticles have the ability to be administered via non-invasive
routes and at the same time achieve effective tumor targeting,
which in turn will reduce the off-target effects and unwanted
immunological reactions and toxicity.
[0168] Evaluation of percentage gene knockdown of PLK1 gene,
silenced by siRNA delivered systemically via novel
receptor-targeted nanoparticles. The PLK1 gene knockdown was
evaluated by extracting total RNA form the tumor tissues, then
reverse transcribing it to form a cDNA and then running a Real
timer PCR on cDNA using primers specific for PLK 1 gene. The
percentage gene knockdown of PLK1 gene by siRNA delivered through
novel receptor-targeted nanoparticles was evaluated by comparing
the treatment group with other controls; mock transfections and
untreated. As represented in FIG. 23, it was observed that the
treatment group showed a significant reduction (P=0.031) in PLK1
gene knockdown of 50% as compared with the untreated control (n=6).
There was no significant difference in the group of animals that
received mock transfections i.e. nanoparticles with NT siRNA
(P=0.933) and PLK1 siRNA alone (P=0.539), when compared with the
untreated control. This result confirmed that the nanoparticles
were able to safeguard the siRNA when administered via systemic
route and delivered it specifically at the tumor site. Under these
experimental conditions, the siRNA retained its functional ability
of gene knockdown and was not degraded by the serum proteins.
[0169] Evaluation of protein suppression of PLK1 protein by siRNA,
delivered systemically via novel receptor-targeted nanoparticles.
To evaluate and compare the amount of protein expression in various
treatment groups, the total protein was extracted from the tumor
tissue, quantified using BCA assay and 100 .mu.g of total protein
was loaded onto NuPAGE.RTM. 4 to 12% Bis Tris gels for western blot
analysis. The gel was electrophoretically transferred to a
nitrocellulose membrane and probed with appropriate antibodies. The
protein bands developed using autoluminiscence were quantified
using an Image J software and plotted with animal numbers, n=6 in
each group. The relative protein expression as observed in FIG. 24
shows that the animals receiving novel receptor-targeted
nanoparticle formulation carrying siRNA against PLK1 gene has
significantly lower protein expression (P=0.038) as compared to the
control, untreated group. However, no difference was observed among
the mock transfection groups i.e. nanoparticles with NT siRNA
(P=0.999) and PLK1 siRNA alone (P=0.758) when compared with the
control untreated group. These results suggest that the siRNA
targeted against PLK1 was solely responsible for the decrease in
the protein expression in the tumor tissue and there was no
deleterious effect posed by the polymeric nanoparticle formulation
on the tumor tissue.
[0170] Serum analysis for safety and toxicity study. 200 .mu.l of
blood was collected in microtainer serum separating tubes just
before the experimental end point, from jugular vein of all the
animals in each group. The experimental end-point was at 2 weeks
after the commencement of the treatment formulation. The serum was
analyzed for safety tests especially for liver function tests
(AST/ALT) and C-reactive proteins (CRP) as represented in FIGS. 25A
and 25B respectively. No significant difference was observed among
any treatment groups when compared to the untreated control for
both CRP and AST/ALT ratios. Thus, these results indicate that the
receptor-targeted nanoparticle formulation was not toxic and does
not stimulate immunological reactions. It also shows that the
percentage gene knockdown and protein suppression as observed in
FIGS. 23 and 24 is the result of gene knockdown mediated by the
siRNA targeted against PLK1 gene leading to cell apoptosis and was
not due to any deleterious/toxic effects of the polymeric
nanoparticle formulation used as a transfection reagent.
[0171] The results presented herein show the potential of novel
receptor-targeted nanoparticle formulation to be used in vivo in a
mouse xenograft model of colon cancer. These nanoparticles prepared
from a chemically modified polymer have the ability to be modified
by incorporation of a specific peptide or a cell targeting
ligand/moiety, which can target a specific receptor on a cell. The
nanoparticles prepared showed efficient siRNA complexation and
delivery with approximately 100% transfection efficiency. The
nanoparticles safe guarded the siRNA when administered systemically
and specifically released it at the tumor site. 50% of gene
knockdown of PLK1 was achieved as determined by qPCR analysis with
siRNA delivered through novel receptor-targeted nanoparticles.
These nanoparticles did not induce any immunological reactions and
liver toxicity as determined by the serum analysis. This study
proves that nanoparticles mediated gene therapy can be achieved and
performed via non-invasive strategies. These nanoparticles as a
delivery device can further be explored towards different
applications such as cancer and neurological disorders. This study
can further be improved by prolonging the treatment duration and
increasing animal number per group in order to achieve a
significant difference and refined interpretation of the data.
EXAMPLE VI
Subject Study for Treating Cancer
[0172] Overexpression of Plk1 has been observed in a number of
human cancers including non-small-cell lung cancer, head and neck
cancer, esophageal cancer, gastric cancer, melanomas, breast
cancer, ovarian cancer, endometrial cancer, colorectal cancer,
glioma, papillary carcinoma, pancreatic cancer, prostate cancer,
hepatoma, leukemia and lymphoma, bladder cancer, and thyroid
cancer. Many of these studies have demonstrated that Plk1
overexpression correlates with tumor progression and patient
survival rate in a variety of cancers. Therefore, Plk1 is proposed
as a prognostic marker for human cancers.
[0173] A subject study was performed on a patient with Stage IV
head and neck cancer. For this study, the synthesis of polymer was
scaled up to obtain 2 g of synthesized polymer, which was used to
form siRNA nanoparticles for the treatment of cancer. The siRNA
used was against PLK1 mRNA, which is a proto-oncogene. The therapy
was termed "Nora-PLK1".
[0174] The scaling up procedure to synthesize the polymer was
performed under aseptic conditions following Good Laboratory
Practices (GLP). Following is the Standard Operating Procedure
(SOP) as detailed herein, [0175] 1) 4 g of Chitosan LMW with 11.04
g of phthalic anhydride in 80 ml of DMF. Stirred for 8 hrs at
110.degree. C. under N.sub.2 atmosphere. Precipitate it in ice cold
water and give a methanol wash overnight. Vacuum filter next
morning using a whatman filter paper on a funnel and vacuum dry.
The polymer will appear light brown in color). Product "c" in FIG.
1. (Phthalic anhydride should be 3 times mole excess of chitosan)
[0176] 2) 60 g of PEG (M.W. 2000) dissolved in 200 ml toluene with
12 g of succinic anhydride dissolved in 40 ml of pyridine. Stir and
reflux the reaction at 100.degree. C. for 6 hrs. Precipitate with
diethyl ether and vacuum filter using a whatman filter paper on a
funnel. Dissolve the product back with chloroform and again
precipitate with diethylether, followed by vacuum filter on a
whatman filter paper using a funnel. Repeat this process twice. At
the end vacuum dry the product. (The product will appear white in
color). Product "a" in FIG. 1. (Succinic anhydride should be 4M
excess of PEG). (Note: Repeat this step in batches for scale up if
required). [0177] 3) To the dried PEG from step 2, dissolve it in
200 ml of toluene and add equimolar of thionyl chloride. Stir and
reflux the reaction at 100.degree. C. for 6 hrs, followed by
degassing to remove excess SO.sub.2 and thionyl chloride. Product
"b" in FIG. 1. [0178] 4) After 6 hrs, from step 3, bring down the
reaction to room temperature (RT) and add phthaloyl chitosan from
step 1 (pre dissolved overnight in pyridine). 1.8 g of chitosan
(pre-dissolved in 50 ml of pyridine) was reacted with 60 g of
PEG2000 (predissolved in 200 ml of toluene). (As the chitosan
(dissolved in pyridine) is added to the PEG (toluene) solution. The
reaction will look heterogeneous). [0179] 5) Let the reaction
continue for 2 hrs at RT and then reflux at 100.degree. C. for 24
hrs. After 24 hrs, bring the reaction down to RT and precipitate in
methanol and vacuum filter dry on a whatman filter paper using
funnel. (The product will appear pale brown in color). Product "d"
in FIG. 1. (PEG2000 should be 10M excess of phthaloyl chitosan).
[0180] 6) The filtered product from step 4, mix 3 g of PEGylated
phthaloyl chitosan with 174 mg of Aluminum chloride in 80 ml of
ethanethiol. Let the reaction stir at 23-25.degree. C. for 12 hrs.
After the reaction, dilute it with water and acidify with 10% HCl.
Vacuum Filter the product on a whatman filter paper using funnel.
Mix the resultant product with deionized water transfer it to a
separating funnel. Extract the purified product in the separating
funnel using dichloromethane three times. Rotavap the
dichloromethane and obtain the dried product. (The product will
appear brown and sticky). Product "e" in FIG. 1 [0181] 7) To the
dried product from step 5, 2.6 g of PEGylated chitosan was
dissolved in 100 ml of toluene and mixed with 452.35 mg of succinic
anhydride (predissolved in 5 ml of pyridine). The reaction is
stirred at 100.degree. C. for 12 hrs. After the reaction the
product brought down to RT and is precipitated in methanol and
vacuum filtered (whatman paper and funnel) and vacuum dry. (The
polymer will appear dark brown solid in color). Product "f" in FIG.
1. (Succinic anhydride should be 4M excess of PEGylated chitosan).
[0182] 8) Take 1 g of the dried CS-PEG-COOH from step 6 and
dissolve it in 25 ml of DMF. Add 1.3 mole equivalent of EDC.HCl to
it (87.5 mg) and 0.1 mole equivalent of DMAP (5.3 mg). Dissolve 500
mg of TAT peptide in 5 ml of DMF and add it to the above mixture.
Stir it for 24 hrs at room temperature. Precipitate the mixture in
ice cold water and filter (whatman paper and funnel), give the
filtrate a brief wash in methanol and again filter dry it. (The
product will appear brown in color). Product "g" in FIG. 1. [0183]
9) Take 1 g of the dried product from step 7 and dissolve it in 20
ml of DMF and dissolve at 80.degree. C. under inert conditions,
after that add 2.6 ml of hydrazine monohydrate to it and let the
reaction stir at similar conditions for 2 hrs. (The product will
appear pale yellow-white in color). Product "h" in FIG. 1. Bring
the reaction to room temperature and Dialyze the product using
slide-a-lyzer (dialysis tube) MW cut off of 3,500 for 2-3 days in
sterilized water. While doing dialysis change the water at least
3-4 times a day. After dialysis, concentrate the product to remove
excess water by vacuum filter (whatman paper and funnel) and/or
freeze dry the sample, overnight and you will obtain an off-white
colored fluffy polymer. [0184] 10) To prepare Nora-PLK1
nanoparticles, dissolve the synthesized Lyophilized polymer at a
concentration of (0.5 mg/ml) in a sterilized water, containing 2%
acetic acid and adjust the pH to 5 using 5M NaOH solution. Stir
this solution overnight at 50.degree. C. Filter the polymer
solution using sterile stericups (filters) with pore size of 0.2
.mu.m. [0185] 11) Mix 2 .mu.g of siRNA (20 .mu.M siRNA stock) to
the polymer (0.5 mg/ml) solution. [To prepare a dose of 50 .mu.g of
siRNA. Aliquout 25 ml of filtered chitosan polymer solution in a
falcon tube and add 195 .mu.l of (20.mu.M siRNA stock) and vortex
vigorously for 5-10 seconds and keep at bench for 30 minutes for
complexation and stabilization. [0186] 12) The nanoparticles after
complexation with siRNA, be filtered through a sterile 0.45 .mu.m
filters. The sterile filtered nanoparticles should be concentrated
using Amicon centrifugal concentrators (MW. Cut-off of 30,000) and
spun down at 4000 rpm for 30-40 minutes to obtain a concentrate of
1.5 to 2 ml volume. (While concentrating care must be taken that
the nanoparticles do not precipitate or aggregate). [0187] 13) For
freeze-drying, the concentrated siRNA-nanoparticles should be
transferred to autoclaved eppendorf tubes and 300 .mu.l of 60%
sucrose solution should be added. The concentrated
siRNA-nanoparticle solution, containing sucrose should be stored in
-20.degree. C. for 30 minutes, followed by freezing in -80.degree.
C. for 1-2 hrs and be placed in a lyophilizer for overnight drying.
After lyophilisation, the siRNA-nanoparticle material can be stored
at -80.degree. C. until use or can be reconstituted in sterilized
water up to 0.5 to 1 ml for delivery (intra tumor or intra venous)
purposes. [0188] 14) The synthesis proved to be reproducible after
being scaled up. The average nanoparticle size was <200 nm with
a zeta potential of 16-20 mV.
[0189] The subject study was performed on female, aged 55 years
old. The patient had Stage IV head and neck cancer, specifically
called squamous cell carcinoma of tonsil. The patient had
sub-mandibular lymph node metastasis and smaller in the right neck.
There are 3 small metastasis in as many left ribs and multiple lung
metastasis in both lungs. The patient had no prior treatment with
chemo or radio therapy. The major outcome of the study was based on
the reduction in the size of the tumors based on the CT scans. The
intervention used for this study included TAT peptide-tagged
PEGylated chitosan nanoparticle carrying PLK1 siRNA, termed as
"Nora-PLK1" therapy.
[0190] The intervention included a total dose of 100 .mu.g (siRNA)
of Nora-PLK1 therapy, administered locally via intra-tumoral
injections at alternate sites in the neck and tongue and/or via
inhalation using a nebulizer/inhaler to reach lungs. The Nora-PLK1
therapy was initiated on Sep. 28, 2014 and was given for a total of
6 weeks, everyday excluding weekends.
[0191] The intratumoral application resulted in a dramatic
reduction of the tumor mass. Volumetrically the tumor reduced from
29.66.times.41.66.times.46 mm to 18.66.times.35.55.times.41 mm or
from 56,839.2 m.sup.3 to 27,197.8 m.sup.3 (a 46% reduction) for the
first 10 day therapy cycle of the Nora-PLK1 treatment (FIG. 26 and
FIG. 27). Followed by complete disappearance of tumor mass by
December 2014.
[0192] Complete disappearance of the tumor mass from lungs was
observed (CT scans) with Nora-PLK1 therapy administered via
inhalation using a nebulizer (FIG. 28).
[0193] The successful results obtained from the subject study
showed, improved health benefits such as, disappearance of cancer
from neck and lungs, improved speech, ability to eat and swallow
food, gain in body-weight. The therapy worked without the need of
any invasive techniques such as surgery or radio/chemotherapy. The
present invention represents a platform technology, wherein the
peptide used in the polymeric synthesis can be replaced with any
other cell-targeting peptide, protein, antibiotic, antibody,
pharmaceutical compounds. The therapeutic payload, complexed with
the nanoparticle is not limited to siRNA, but can include, pDNA,
miRNA, small oligonucleotides, chemotherapeutic drugs,
nutraceutical compounds. The nanoparticles produced can also be
formulated in a hydrogel for topical application purposes, or mixed
with microparticles for oral delivery purposes to provide a varied
range of its health beneficial effects.
[0194] While the invention has been described in connection with
specific embodiments thereof, it will be understood that it is
capable of further modifications and this application is intended
to cover any variations, uses, or adaptations of the invention
following, in general, the principles of the invention and
including such departures from the present disclosure as come
within known or customary practice within the art to which the
invention pertains and as may be applied to the essential features
herein before set forth, and as follows in the scope of the
appended claims.
REFERENCES
[0195] Calvo, P, Remunan-Lopez, C, Vila-Jato, J L, Alonso, M J:
Novel hydrophilic chitosan-polyethylene oxide nanoparticles as
protein carriers. J Appl Polym Sci 63:125-132, 1997. [0196] Kurita
K, Ikeda H, Yoshida Y, Shimojoh M, Harata M: Chemoselective
protection of the amino groups of chitosan by controlled
phthaloylation: facile preparation of a precursor useful for
chemical modifications. Biomacromolecules 3:1-4, 2002.
[0197] Lin W J, Chen M H: Synthesis of multifunctional chitosan
with galactose as a targeting ligand for glycoprotein receptor.
Carbohydrate polymers 67:474-480, 2007.
[0198] Malhotra, M, Kulamarva, A, Sebak, S, Paul, A, Bhathena, J,
Mirzaei, M, Prakash, S: Ultrafine chitosan nanoparticles as an
efficient nucleic acid delivery system targeting neuronal cells.
Drug Development and Industrial Pharmacy 35:719-726, 2009.
Sequence CWU 1
1
15124PRTArtificial SequenceMGF peptide 1Tyr Gln Pro Pro Ser Thr Asn
Lys Asn Thr Lys Ser Gln Arg Arg Lys 1 5 10 15 Gly Ser Thr Phe Glu
Glu His Lys 20 27PRTArtificial SequenceCP15 peptide 2Val His Leu
Gly Tyr Ala Thr 1 5 312PRTArtificial SequenceP160 peptide 3Val Pro
Trp Met Glu Pro Ala Tyr Gln Arg Phe Leu 1 5 10 49PRTArtificial
SequenceTAT peptide 4Arg Lys Lys Arg Arg Gln Arg Arg Arg 1 5
521RNAArtificial SequencePSN1 siRNA 5aagguccacu ucguaugcug g
21618DNAArtificial SequenceOligonucleotide 6ccgaaatcac agccaaga
18722DNAArtificial SequenceOligonucleotide 7cattcacaga agataccaag
ac 22823DNAArtificial SequenceOligonucleotide 8taaagggcat
cctgggctac act 23923DNAArtificial SequenceOligonucleotide
9ttactccttg gaggccatgt agg 231021RNAArtificial SequencePLK siRNA
sensemodified_base(4)..(4)ummodified_base(10)..(10)ummodified_base(14)..(-
14)ummodified_base(18)..(18)um 10agaucacccu ccuuaaauau u
211121RNAArtificial SequencePLK siRNA
antisensemodified_base(8)..(8)gmmodified_base(17)..(17)um
11uauuuaagga gggugaucuu u 211220DNAArtificial
SequenceOligonucleotide 12ggcaaccttt tcctgaatga 201320DNAArtificial
SequenceOligonucleotide 13aatggaccac acatccacct 201423DNAArtificial
SequenceOligonucleotide 14taaagggcat cctgggctac act
231523DNAArtificial SequenceOligonucleotide 15ttactccttg gaggccatgt
agg 23
* * * * *