DESC:F O R M 2
THE PATENTS ACT, 1970 (39 of 1970)
&
THE PATENTS RULES, 2003
PROVISIONAL SPECIFICATION
[See section 10 and rule 13]
1. TITLE OF THE INVENTION: NANOPARTICLE BASED DRUG
DELIVERY SYSTEM TO TARGET
CANCER CELLS
2. APPLICANT (A) Mazumdar Shaw Medical Foundation
(B) Mazumdar Shaw Medical Foundation
A-Block, 8th Floor, Mazumdar Shaw Medical Centre,
#258/A, Narayana Health City, Bommasandra,
Bangalore, Karnataka, India, 560099
3. NATIONALITY (C) INDIA
THE FOLLOWING SPECIFICATION PARTICULARLY DESCRIBES THE NATURE OF THIS
INVENTION AND THE MANNER IN WHICH IT IS TO BE PERFORMED
Page 2 of 21 LI1011-MSMF-001-PR- Provisional
TECHNICAL FIELD OF THE INVENTION
[001] The present invention is in the technical field of nanoparticle delivery system,
in particular, a self-assembling reconstituted high density lipoprotein complex comprising
a combination of: a pH-specific polymer, a cross linker, a receptor binding component (R),
and a drug (D); wherein the pH-specific polymer crosslinked to receptor binding
component (R) and Drug (D), encapsulated within the nano matrix, for targeted and
controlled release of drug for cancer treatment. The present invention also relates to
components useful in preparation of the nanoparticle delivery system, as well as the
compositions, methods, processes, and uses thereof.
BACKGROUND OF THE INVENTION
[002] Cancer cells commonly develop defense mechanisms against the treatment
during the course of chemotherapy, leading to therapy failure and tumor relapses. The
acquisition of cancer drug resistance can be attributed to inefficient drug delivery to tumor
tissues and tumor cells, which results in low drug concentrations at the tumor sites and
incomplete treatment.
[003] In addition, anticancer drugs are typically toxic towards both healthy
proliferating cells as well cancer cells, drug dosage must be restricted to avoid potentially
lethal side effects. Therapeutic efficacy of such restricted drug dosage is further
diminished by factors such as poor pharmacokinetics of the drugs, including limited
systemic circulation lifetime, undesirable bio distribution and non-specific cellular uptake,
and poor tumor vascularity that limits drug accessing to tumor tissues.
[004] Furthermore, the chemotherapy is performed by introducing the raw drug
intravenously to the patient. But this will lead to systemic toxicity and results in
undesirable side effects.
[005] Selective augmentation of anticancer drug concentrations within tumor tissues
remains a major challenge in improving therapeutic efficacy with minimal side effects.
The efficient release of the therapeutic agents (drugs) at the target site is a prerequisite for
effective therapy.
[006] At present, controlled release of drug by use of nanotechnology to a patient
with active ingredient has been an important area of research. These nanocarriers have
demonstrated desirable drug delivery characteristics such as prolonged systemic
circulation lifetime, reduced non-specific cellular uptake, targeting abilities, controllable
drug release, and multidrug encapsulation for combinatorial treatment while minimizing
undesirable side effects.
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[007] It may be appreciated that pH-sensitive polymeric nanoparticles that are capable of
retaining drug during circulation while actively releasing it at the tumor site and/or inside
the target tumor cells have received an overwhelming interest for tumor-targeting cancer
chemotherapy. This smart delivery approach can efficiently resolve the in vivo stability
versus intracellular drug release problem, as well as stealth versus tumor cell uptake
issues. As a result of the active metabolism of tumor cells, the tumor microenvironment
(TME) is highly acidic compared to normal tissues. pH-sensitive nano-systems have now
been focused in which drug release is specifically triggered by the acidic tumor
environment.
[008] In the present scenario, there are challenges to have a nonanoparticle drug delivery
system which is less than 100 nm with successful conjugation and effective encapsulation
as well as combined pH-triggered, receptor-mediated specific drug release and enhanced
therapeutic index with minimal systemic toxicity.
[009] Furthermore, there is also need for a nanoparticulate system that can be made widely
useful by using different receptors/ligands specific and effective to different types cancer.
[010] In summary, existing nanoparticulate systems have challenges in terms of size of
nanoparticle, effective encapsulation, efficient drug release and also having the options of
different combinations of receptors and ligands for treatment different types of cancer.
There is also a need for nanoparticulate systems to address the issue of tumor
heterogeneity.
[011] Accordingly, there is an urgent need for a multifunctional pH responsive nanoparticle
drug delivery system for both hydrophobic and hydrophilic cancer drugs in cancer therapy
with size less than 100 nm with the option of having variable receptor-ligand combination
with successful conjugation, effective encapsulation and enhanced therapeutic index with
minimized systemic toxicity, that can be used in combinatorial chemotherapy.
SUMMARY OF THE INVENTION
[012] According to an exemplary aspect, the present invention discloses a nanoparticle
delivery system, in particular, a self-assembling reconstituted high density lipoprotein
complex comprising a combination of : a pH-specific polymer, a cross linker, a receptor
binding component (R), and a drug (D); wherein the pH-specific polymer crosslinked to
receptor binding component (R) and Drug (D), encapsulated within the nano matrix, for
targeted and controlled release of drug for cancer treatment. The present invention also
relates to components useful in preparation of the nanoparticle delivery system, as well as
the compositions, methods, processes, and uses thereof.
Page 4 of 21 LI1011-MSMF-001-PR- Provisional
[013] Yet another exemplary aspect of the present invention, the nanoparticulate system, the
Chitosan-receptor (R) crosslinked nanoparticles deliver anti-cancer drug (D) for cancer
treatment. The proposed system (Chitosan-R)-D nanocomplex, where R is the receptor for
ligands expressed specifically by cancer cells and D is the anti-cancer drug. The pH
sensitive polymer chitosan is crosslinked to receptor (R) and Drug (D) is encapsulated
within the nano matrix.
[014] According to a further exemplary aspect of the present invention, effective
conjugation (crosslinking) of chitosan to receptor results in higher percentage of receptor
to be exposed to outside and also necessary for higher drug encapsulation and sustainable
drug release (controlled manner).
[015] Yet another exemplary aspect of the present invention, this pH-sensitive nano-system
releases drug specifically triggered by the acidic tumor environment (at specific
endosomal pH conditions).
[016] According to a further exemplary aspect of the present invention, this nanoparticle
delivery system is made more specific by targeting cancer cell-specific receptor by using
particular ligands. Inventors have used HA (Hyaluronic acid) to specifically target breast
carcinoma where, HA can bind to CD44 receptor, highly expressed in cancer cells.
Additionally, this nanoparticulate system can be made widely useful by using different
receptors/ligands specific to different cancer. Chitosan/ligands can be functionalized with
different chemical groups to make this system even more specific and effective.
[017] According to a further exemplary aspect of the present invention, encapsulating the
drug in nanoparticle system will increase systemic life-span of the drug with minimal side
effects and less systemic toxicity.
[018] In summary, the present invention discloses a nanoparticle delivery system, in
particular, a self-assembling reconstituted high density lipoprotein complex comprising a
combination of : a pH-specific polymer, a cross linker, a receptor binding component (R),
and a drug (D); wherein the pH-specific polymer crosslinked to receptor binding
component (R) and Drug (D), encapsulated within the nano matrix, for targeted and
controlled release of drug for cancer treatment with enhanced encapsulation and effective
conjugation, more specific by targeting cancer cell-specific receptor by using particular
ligands. Chitosan/ligands can be functionalized with different chemical groups to make
this system even more specific and effective.
Page 5 of 21 LI1011-MSMF-001-PR- Provisional
[019] The present invention also relates to components useful in preparation of the
nanoparticulate drug delivery system, as well as the compositions, methods, processes, and
uses thereof.
[020] Several aspects of the invention are described below with reference to examples for
illustration. However, one skilled in the relevant art will recognize that the invention can
be practiced without one or more of the specific details or with other methods,
components, materials and so forth. In other instances, well-known structures, materials,
or operations are not shown in detail to avoid obscuring the features of the invention.
Furthermore, the features/aspects described can be practiced in various combinations,
though only some of the combinations are described herein for conciseness.
[021] As will be appreciated by a person skilled in the art the present invention provides a
variety of following embodiments. Listing of Claims,
1) A nanoparticle delivery system, comprising: a self-assembling reconstituted high density
lipoprotein complex comprising a combination of:
a) a pH-specific polymer;
b) a cross linker;
c) a receptor binding component (R); and
d) a drug (D)
wherein the pH-specific polymer crosslinked to receptor binding component (R) and Drug
(D) encapsulated within the nano matrix, and the self-assembling reconstituted high
density lipoprotein complex has a diameter in the range of from about 70 nm to 110 nm.
2) The nanoparticle delivery system of [021.1], wherein the pH-specific polymer comprises a
chitosan component.
3) The nanoparticle delivery system of [021.1], wherein the system is a specific combination
of a cancer cell-specific receptor and a particular ligand.
4) The nanoparticle delivery system of [021.1], wherein specific receptors/ligands are
functionalized with specific chemical groups to make this system specific and effective for
a specific cancer type.
5) The nanoparticle delivery system of [021.1], wherein the nanoparticle delivery vehicle is
spherical, oval, or discoidal in shape.
6) The nanoparticle delivery system of [021.1], wherein the nanoparticle delivery vehicle
further comprises a pharmaceutical agent.
7) The nanoparticle delivery system of [021.6], wherein the pharmaceutical agent is located
in the core of the nanoparticle delivery vehicle.
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8) The nanoparticle delivery system of [021.6], wherein the pharmaceutical agent is a
lipophilic.
9) The nanoparticle delivery system of [021.6], wherein the pharmaceutical agent is a
hydrophilic.
10) The nanoparticle delivery system of [021.1], wherein the core of the nanoparticle
represents a lipophilic and nonaqueous environment.
11) The nanoparticle delivery system of [021.1], wherein the lipid binding protein component
is modified to enhance the targeting efficacy of the drug.
12) The nanoparticle delivery system of [021.1], wherein the lipid binding protein component
is modified by the attachment of antibodies, folic acid residues or other ligands that target
the surface of malignant cells and tumors.
13) The nanoparticle delivery system of [021.1], further comprising a functional moiety which
augments the efficacy of the pharmaceutical agent.
14) The nanoparticle delivery system of [021.13], wherein the functional moiety targets the
nanoparticle delivery system through interaction with a cell surface receptor.
15) A method for delivering a drug of interest to a subject, comprising: administering the
nanoparticle delivery system of [021.6], to the subject; wherein the pharmaceutical agent
is the drug of interest for cancer.
16) A method of treating cancer in a subject, comprising: administering the nanoparticle
delivery system of [021.6] to the subject; wherein the pharmaceutical agent is a
chemotherapeutic agent.
17) The method of [021.15], wherein the nanoparticle delivery system is delivered
intravenously, subcutaneously, parenterally, intramuscularly, transdermally or
transmucosally,
18) The method of [021.15], wherein the cancer is breast cancer.
Page 7 of 21 LI1011-MSMF-001-PR- Provisional
BRIEF DESCRIPTION OF THE DRAWINGS
[022] Example embodiments of the present invention will be described with reference to
the accompanying drawings briefly described below.
[023] FIG. 1 illustrates a schematic mechanism of pH-responsive release behaviour of the
chitosan NPs according to aspects of the present invention. The anticancer drug is
represented by red rectangles.
[024] FIG. 2 illustrates a schematic illustration of proposed combined Nano delivery
system; (Chitosan-HA)-Paclitaxel nanoparticles for receptor mediated pH specific drug
release according to aspects of the present invention.
[025] FIG. 3 illustrates an example of the formation of stable conjugate (amide bond) by
crosslinking chitosan NPs with HA by carbodiimide reaction with EDC and NHS as
catalysis according to aspects of the present invention.
[026] FIG. 4 graphs illustrating the size distribution pattern of chitosan NPs, Chitosan-HA
NPs, Chitosan-HA-Paclitaxel NPs determined by dynamic light scattering (DLS) method
after optimizing synthesis and conjugation steps of Chitosan-HA-Paclitaxel NPs
according to aspects of the present invention.
[027] FIG. 5 graphs illustrating the size distribution pattern of chitosan NPs, Chitosan-HA
NPs, Chitosan-HA-Paclitaxel NPs determined by dynamic light scattering (DLS) method
after modifying standard protocols according to aspects of the present invention.
[028] FIG. 6 graph illustrating the Charge distribution pattern of chitosan NPs, Chitosan-
HA NPs, Chitosan-HA-Paclitaxel NPs determined by dynamic light scattering (DLS)
method according to aspects of the present invention.
[029] FIG. 7 graph illustrates the Size distribution pattern of chitosan NPs, Chitosan-HA
NPs, Chitosan-HA-Dox NPs determined by dynamic light scattering (DLS) method after
modifying standard protocols according to aspects of the present invention.
[030] FIG. 8 graph illustrates the In vitro Paclitaxel (Paclitaxel) release from Chitosan-HAPaclitaxel
NPs according to aspects of the present invention.
[031] FIG. 9 illustrates the In vitro Doxorubicin (Dox) release from Chitosan-HA-Dox NPs
according to aspects of the present invention.
[032] FIG. 10 illustrates the ATR-FTIR spectrum of Chitosan-HA NPs and Chitosan-HAPaclitaxel
NPs Relative composition of HA and Chitosan in Chitosan-HA nanoparticles.
[033] FIG 11 shows graph for the relative composition of HA and Chitosan in Chitosan-HA
nanoparticles (Spectrophotometric method).
Page 8 of 21 LI1011-MSMF-001-PR- Provisional
[034] FIG. 12 illustrates the Fe-SEM pictographs of Chitosan-HA NPs and Chitosan-HADox
NPs were investigated by Field emission scanning electron microscopy.
[035] FIG 13 shows the Dose-response curves for MDAMB 231, HaCaT, HGF and NIH3T3
following treatment with Drug (Paclitaxel), HA (Hyaluronic acid) NPs, HA-CN
(Hyaluronic acid-Chitosan) NPs and HA-CN-Paclitaxel (Hyaluronic acid-Chitosan-
Paclitaxel) NPs at 48 hours.
[036] FIG. 14 illustrates the graph for Cell viability of MDA-MB-231, HaCaT, NIH 3T3
after treatment with different nanoformulations (Dox: Doxorubicin (control), HC Dox:
Chitosan-HA-Doxorubicin NPs) after 48 hr and 72 hr. Error bar represents standard
deviation of mean; *** indicates p<0.001, ** indicates p<0.01, * indicates p<0.05.
[037] FIG. 15 illustrates the Cell viability of MDA-MB-231, HaCaT, NIH 3T3 after
treatment with different nanoformulations (Dox: Doxorubicin (control), HC Dox:
Chitosan-HA-Doxorubicin NPs, CN Dox: Chitosan-Doxorubicin NPs, HA-Dox: HA
Doxorubicin NPs) after 48 hr and 72 hr. Error bar represents standard deviation of mean;
*** indicates p<0.001, **indicates p<0.01, *indicates p<0.05.
[038] In the drawings, like reference numbers generally indicate identical, functionally
similar, and/or structurally similar elements. The drawing in which an element first
appears is indicated by the leftmost digit(s) in the corresponding reference number.
DETAILED DESCRIPTION OF THE INVENTION
[039] It is to be understood that the present disclosure is not limited in its
application to the details of construction and the arrangement of components set forth in
the following description or illustrated in the drawings. The present disclosure is capable
of other embodiments and of being practiced or of being carried out in various ways. Also,
it is to be understood that the phraseology and terminology herein is for the purpose of
description and should not be regarded as limiting.
[040] The use of “including”, “comprising” or “having” and variations thereof herein is
meant to encompass the items listed thereafter and equivalents thereof as well as
additional items. The terms “a” and “an” herein do not denote a limitation of quantity, but
rather denote the presence of at least one of the referenced item. Further, the use of terms
“first”, “second”, and “third”, and the like, herein do not denote any order, quantity, or
importance, but rather are used to distinguish one element from another.
[041] All documents cited in the present specification are hereby incorporated by reference
in their totality. In particular, the teachings of all documents herein specifically referred to
are incorporated by reference.
Page 9 of 21 LI1011-MSMF-001-PR- Provisional
[042] The pharmaceutical formulations of the said invention may also include
pharmaceutically acceptable nontoxic carriers or diluents, for animal or human
administration.
[043] The present invention concerns methods and compounds useful for the protection and
treatment of proliferative and/or inflammatory disorders.
[044] “Proliferative disease or disorder” means all neoplastic cell growth and proliferation,
whether malignant or benign, including all transformed cells and tissues and all cancerous
cells and tissues. Proliferative diseases or disorders include, but are not limited to,
premalignant or precancerous lesions, abnormal cell growths and/or lesions, benign
tumours, malignant tumours, and “cancer.”
[045] Additional examples of proliferative diseases and/or disorders include, but are not
limited to neoplasms, whether benign or malignant, located in the: liver, pancreas,
peritoneum, endocrine glands (adrenal, testicles, ovary, thymus, thyroid, parathyroid,
pituitary), eye, head and neck, nervous (central and peripheral), soft tissue, spleen,
thoracic, lymphatic system, pelvic, skin, and urogenital tract embodiment. According to a
non limiting exemplary aspect of the present invention, the proliferative disorder involves
tumour.
[046] The terms “tumour” or “tumour tissue” refer to an abnormal mass of tissue which
results from excessive cell division. A tumour or tumour tissue comprises “tumour cells”
which are neoplastic cells with abnormal growth properties and no useful bodily function.
[047] Tumours, tumour tissue and tumour cells may be benign or malignant. A tumour or
tumour tissue may also comprise “tumour-associated non-tumour cells”, e.g., vascular
cells which form blood vessels to supply the tumour or tumour tissue. Non-tumour cells
may be induced to replicate and develop by tumour cells, for example, the induction of
angiogenesis in a tumour or tumour tissue. According another non limiting exemplary
aspect of the present invention, the proliferative disorder involves malignancy or cancer.
[048] Example embodiments of the present invention are described with reference to the
accompanying figures.
[049] In the drawings, like reference numbers generally indicate identical, functionally
similar, and/or structurally similar elements. The drawing in which an element first
appears is indicated by the leftmost digit(s) in the corresponding reference number.
[050] The present invention discloses a nanoparticulate drug delivery system, in particular,
the system includes pH-specific polymer (chitosan) crosslinked to receptor (R) for ligands
expressed by cancer cells and drug (D) is encapsulated within the nano matrix, which
Page 10 of 21 LI1011-MSMF-001-PR- Provisional
offers a novel delivery system with enhanced encapsulation and effective conjugation,
with reduced the size of the combined nanocomplex. The nanoparticulate system can be
made more specific by using variable combinations of cancer cell-specific receptor and
ligands. The term “Chitosan”, is meant to refer N-deacetylated chitin and other watersoluble
derivatives of chitin.
IMPORTANT EMBODIMENTS OF THE SYSTEM:
[051] 1. Effective conjugation: Effective conjugation (crosslinking) of chitosan to receptor
is achieved, so that higher percentages of receptors are exposed to outside which is
essential for higher drug encapsulation and sustainable drug release (controlled manner).
[052] 2. pH responsive drug release: As a result of active metabolism of tumor cells, the
tumor microenvironment (TME) is highly acidic compared to normal tissues. pH-sensitive
chitosan absorb protons at endosomal pH (5.2), leading to an increase in osmotic pressure
inside the endosomal compartment, followed by plasma membrane disruption and
polymeric nanoparticle (NP) release into the cytoplasm. The endosomal pH specific drug
release would also escape premature degradation by lysosomal pH as well as degradative
enzymes so that this novel delivery system can effectively make the drug to attain
maximum pharmacological effect.
[053] This system will also contribute for the specificity to the solid tumors, since the tumor
microenvironment is acidic; the drug will be delivered only at the premises of the tumor as
shown in FIG 1.
[054] Specific targeting: This nanoparticle delivery system can be made more specific by
targeting cancer cell specific receptor by using particular ligands.
a. e.g. we have used HA (Hyaluronic acid) to specifically target breast carcinoma where, HA
can bind to CD44 receptor, highly expressed in cancer cells.
b. Similarly, this nanoparticulate system can be made widely useful by using different
receptors/Ligands specific to different cancer.
[055] Minimal side effects: Encapsulating drug into our nanoparticle will increase systemic
life span of the drug and will not affect the non-specific cells, so there will not be any
systemic toxicity or very minimal side effects.
[056] Functionalization of pH responsive polymer/ligands: Chitosan/Ligands can be
functionalized with different chemical groups to make this system more specific and
effective.
Page 11 of 21 LI1011-MSMF-001-PR- Provisional
a. e.g. HA can be modified with CHO-groups (HA-CHO), so that it can be acted upon by the
aldehyde hydrogenase enzyme (highly expressed in breast carcinoma cells) making the
system more specific (by including multiple specificity).
[057] Example embodiments: Proposed nanoplatform to target breast carcinoma:
(Chitosan-HA)-Paclitaxel and Chitosan-HA)-Doxorubicin nanoparticles.
[058] Invention proposes a chitosan-hyaluronic acid nanoparticles to deliver
Paclitaxel/Doxorubicin drug as for breast carcinoma treatment. The chitosan nanoparticle
could act as pH responsive polymer while Hyaluronic acid (HA) could act as breast cancer
specific receptor (hyaluronan, which bears CD44 ligand). The developed nanocomplex
would act as excellent delivery system for hydrophobic cancer drugs such as Paclitaxel, as
described in FIG 2.
[059] Embodiments of the Invention,
[060] Formulation of Functionalized Nanocomposite (Chitosan-HA-Paclitaxel
nanoparticles and Chitosan-HA-Dox nanoparticles)
[061] In vitro Efficiency (Drug)
i. Drug Encapsulation Efficiency study
ii. Drug Release study (in vitro)
iii. Specificity (in cell lines)
[062] Characterization of Functionalized Nanocomposite
i. Dynamic Light Scattering analysis (Size and charge)
ii. Fourier transform infrared spectroscopy (FTIR) study
iii. Surface morphology
iv. Molecular composition
[063] In vivo evaluation of Chitosan-HA-Paclitaxel and Chitosan-HA-Dox nanoparticles.
[064] Significant Results as part of the embodiments of the invention:
[065] 1. Effective conjugation of Chitosan nanoparticles with HA by carbodiimide
reaction with EDC and NHS as catalysis that successfully encapsulated the drug
(Paclitaxel; encapsulation efficiency 80.8% and 32.65% for Doxorubicin) and the
controlled Paclitaxel release was observed from Chitosan-HA-Paclitaxel NPs at pH 5.2
(48.26% at 24 hr, 64.24% at 48 hr, 70.67% at 72 hr) and Dox release from Chitosan-HADox
NPs at pH 5.2 2 (31.2% at 24 hr, 61.24% at 48 hr, 70.67% at 72 hr).
[066] The HA (ligands for the receptor CD44) was exposed to the surface of the
nanocomplex (revealed from zeta potential analysis, FTIR analysis study and relative
composition analysis).
Page 12 of 21 LI1011-MSMF-001-PR- Provisional
[067] In vitro assay confirmed the specificity of this system that avoids systemic toxicity or
very minimal side effects, as Chitosan-HA-Paclitaxel nanoparticles selectively killed only
breast cancer cell line MDA MB 231 and did not affect the normal cell lines HEK 293T
and HaCaT.
[068] 2. Formulation of Functionalized Nanocomposite (Chitosan-HA-Paclitaxel and
Chitosan-HA-Dox nanoparticles), Synthesis of chitosan nanoparticles
[069] Chitosan nanoparticles were prepared via the ionotropic gelation of chitosan with TPP
(Tri poly phosphate) anions according to a modified method of Calvo et al. 1997. LMW
chitosan was dissolved in an aqueous solution of acetic acid (0.5%) to form a 1 mg/mL
chitosan solution. The chitosan solution was stirred (400 rpm) overnight at room
temperature using a magnet stirrer. The resulting chitosan solution was sonicated 30 min at
50W (in ice) and then pH of the resulting solution was adjusted to ~4.5 using sodium
hydroxide solution (10M). The chitosan solution was then filtered through a syringe filter
(pore size 0.45 µm) to remove insoluble particles. The aqueous solution of TTP was
prepared at a concentration of 1 mg/mL and also filtered through a syringe filter (pore size
0.45 µm). 20 ml of chitosan solution was preheated in a water bath at 60?C for 15 min and
then kept on the magnetic stirrer stirring at 800 rpm. 5 ml of chilled (4?C) TPP solution
was added (with a syringe having 24 gauge needle, constant force) to the chitosan solution.
The reaction was carried out for 60 min followed by sonication for 30 min at 50W (in ice),
leaving then the dispersion undisturbed for 12 h prior to further analysis (FIG 3).
[070] 3. Chitosan-HA-Paclitaxel nanoparticles
a. The sodium (Na+) ions were removed from Hyaluronic acid sodium salt by dialysis. 50mg
of HA was dissolved in 50 mL of deionized water and dialyzed (MWCO: 12kD) for 12h in
dilute acid. The synthesis of Chitosan-HA nanoparticle was carried out by carbodiimide
reaction with EDC and NHS as catalysis. The scheme to crosslink between carboxyl group
of HA to amine group of chitosan is specifically designed by the inventors.
[071] 4. Examples embodiments of the formulation of functionalized nanocomposite
(Chitosan-HA-Paclitaxel nanoparticles) and synthesis of chitosan nanoparticles include,
but are not limited to, small molecules, polynucleotides, polypeptides, polysaccharides,
fatty acids, lipids, and antibodies.
[072] Characterization of functionalized nanocomposite: Dynamic Light Scattering
measurements
[073] The size (Hydrodynamic diameter), size distribution (Poly Dispersity Index) and zeta
potential (Surface charge) of the NPs were analyzed by Zeta sizer (ZS 90, Malvern
Page 13 of 21 LI1011-MSMF-001-PR- Provisional
Instruments Ltd, Malvern, UK). The average size of chitosan NPs, Chitosan-HA NPs,
Chitosan-HA-Paclitaxel NPs were 65.92 nm, 175.1 nm and 199.2 nm respectively. The
size is increased in Chitosan-HA-Paclitaxel NPs with addition of HA and Paclitaxel, as
shown in FIG 4.
[074] Although, Nanoparticles are colloidal particles having a size of 10 to 1000 nm, it is
demonstrated that for an ideal targeting carrier the particle should be less or equal to 100
nm. So, we have standardized our protocol (synthesis of chitosan nanoparticle,
conjugation of chitosan with HA) to reduce the size of the nanocomplex without affecting
encapsulation efficiency as well as drug release behavior. The average size of newly
synthesized Chitosan-HA NPs, Chitosan-HA-Paclitaxel NPs were 89.25 nm and 106.9 nm
respectively. Chitosan-HA-Paclitaxel NPs are of the size ~100 nm that would
considerably avoid the RES (reticuloendothelial system) uptake, as described in FIG 5.
[075] The zeta potential indicates the degree of repulsion between adjacent particles and
similarly charged particles in dispersion. For molecules and particles that are small
enough, a high zeta potential confers stability (dispersion will resist aggregation). When
the zeta potential is low, the attraction between the particles exceeds repulsive force for
which leads to flocculate.
[076] The average charge of chitosan NPs, Chitosan-HA NPs, Chitosan-HA-Paclitaxel NPs
were 26.92 mV, -14.8 mV and -15.6 mV respectively. The charge is changed from
positive to negative due to negatively charged HA in Chitosan-HA NPs, Chitosan-HAPaclitaxel
NPs. The negative charge confirms the presence of HA in the Chitosan-HAPaclitaxel
NPs and it likely to be exposed to surface of the nanocomplex, as shown in FIG
6.
[077] The average size of newly synthesized Chitosan-HA NPs, Chitosan-HA-Dox NPs
were 89.6 nm and 110 nm respectively. Chitosan-HA-Dox NPs are of the size ~100 nm
that would considerably avoid the RES (reticuloendothelial system) uptake, as shown in
FIG 7.
[078] Drug content
[079] The drug loading efficiency depends upon the polymer nature, physical state of the
drug and the molecular interactions between the drug and the polymer. Paclitaxel
(Paclitaxel) encapsulation efficiency (%) in Chitosan-HA NPs was 80.8%. Doxorubicin
(Dox) encapsulation efficiency (%) in Chitosan-HA NPs was 32.65±3.5%. So, it is
confirmed that the size of Chitosan-HA-Dox NPs was reduced without affecting drug
loading behavior.
Page 14 of 21 LI1011-MSMF-001-PR- Provisional
[080] Drug Release study (In vitro)
[081] In vitro release of Paclitaxel was assessed from Chitosan-HA NPs into PBS at pH 7.4
and at pH 5.2 to observe the pH responsive drug release. The pH responsive drug release
profile was observed in case of Chitosan-HA-Paclitaxel NPs at pH 5.2.The successful and
controlled Paclitaxel release was observed from Chitosan-HA-Paclitaxel NPs at pH 5.2
(48.26% at 24 hr, 64.24% at 48 hr, 70.67% at 72 hr). Paclitaxel release from Chitosan-
HA-Paclitaxel at pH 7.2 was very less as only 21.42% released after 72 hr.
[082] Similarly, In vitro release of Doxorubicin was assessed from Chitosan-HA NPs into
PBS at pH 7.4 and at pH 5.2 to observe the pH responsive drug release. The pH
responsive drug release profile was observed in case of Chitosan-HA-Dox NPs at pH
5.2.The successful and controlled Dox release was observed from Chitosan-HA-Dox NPs
at pH 5.2 (31.2% at 24 hr, 61.24% at 48 hr, 70.67% at 72 hr).Dox release from Chitosan-
HA-Dox at pH 7.2 was very less as only 21.42% released after 72 hr, as described in FIG
8 and FIG 9.
[083] Surface chemical composition analysis by Fourier Transform Infrared (FTIR)
spectroscopy
[084] FTIR analysis measures the selective absorption of light by the vibration modes of
specific chemical bonds in the sample. The characterization by Fourier Transform Infrared
(FTIR) was specifically carried out to determine the encapsulation of the drug (Paclitaxel
and Doxorubicin) in the Chitosan-HA NPs by studying the chemical properties of
Paclitaxel/Dox encapsulated NPs. There are nearly similar peaks were observed in case of
Chitosan-HA NPs and Chitosan-HA-Paclitaxel/Dox NPs that confirms complete
encapsulation of the drug in to Chitosan-HA nano matrix (also no peaks corresponding to
Paclitaxel/Dox drug were observed). Again, there are nearly similar peaks corresponding
to chitosan molecules were observed [1154.22 cm-1 (COOC bridge), 2814.18 cm-1(C-H
stretching), 1390.78 cm-1(Amide III bonding), 1606.94 cm-1(NH2 boding), 3740.01 cm-1
(O-H stretching) etc.]. There are nearly similar peaks corresponding to Hyaluronic Acid
molecules were observed [811.62 cm-1, 566.35 cm-1 (phosphate group), 1533.52 cm-
1(Amide II bonding), 1684.43 cm-1(Amide I bonding), 3740.01 cm-1 (O-H stretching)
etc.].
[085] Hence, FTIR results confirmed that both chitosan and HA molecules are surface
exposed in the formulated Chitosan-HA-Paclitaxel and Chitosan-HA-Dox nanoparticles as
described in FIG 10.
[086] Relative composition of HA and Chitosan in Chitosan-HA nanoparticles
Page 15 of 21 LI1011-MSMF-001-PR- Provisional
[087] Spectrophotometric method was followed to examine the relative composition of HA
and Chitosan using standard curve method prepared by varying concentration of
Paclitaxel, HA, Chitosan separately. The relative composition of Chitosan, HA, Drug in
Chitosan-HA-Paclitaxel nanoparticles were 39.6 %, 51.5 % and 8.5 % respectively.
[088] The lower amount of drug as reported from spectrophotometric method is due the
encapsulation of the drug within the chitosan-HA matrix. So, there is very less amount of
drug exposed to outside that has been detected, as shown in FIG 11.
[089] Morphological studies by Scanning Electron Microscopy (SEM)
[090] The surface morphologies of Chitosan-HA NPs and Chitosan-HA-Paclitaxel/Dox NPs
were investigated by Field emission scanning electron microscopy (Nova, NanoSEM,
450/FEI). The nanoparticles were fixed on an adequate support and coated with platinum
using platinum sputter module in a higher vacuum evaporator. Observations under
different magnifications were performed at 10 kV.
[091] The Fe-SEM images further confirm the results obtained in DLS study (size
measurement). The Chitosan-HA NPs were found to be roughly spherical in shape with
some rhombic surface modifications, whereas the Chitosan-HA-Paclitaxel/Dox NPs were
found to be smooth spherical in shape with clear shiny texture, as shown in FIG 12.
[092] In vitro cytotoxicity study by MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-
Diphenyltetrazolium Bromide] assay (Chitosan-HA-Paclitaxel NPs)
[093] Breast cancer cell line MDA MB 231 and normal cell lines HaCaT, HGF, NIH3T3
were treated with paclitaxel carrying nanoparticles with respective controls for both 24
hours and 48 hours. Free paclitaxel killed all the 4 types of cell lines i.e. MDA MB 231
(IC 50~ 11.1nM), HaCaT (IC 50~ 1.66 nM), HGF (IC 50~ 2.6 nM), and NIH3T3 ((IC
50~3.061 nM). However, the nanoparticles loaded with paclitaxel killed only breast cancer
cell line MDA MB 231 (IC 50~ 1.978 nM) and did not affect the normal cell lines HaCaT
(IC 50~ 798.7 nM), HGF (IC 50~ 615.2 nM), and NIH3T3 ((IC 50~743.8 nM), as shown
in FIG 10. Hence, it is proved that Chitosan-HA-Paclitaxel nanoparticles selectively killed
only breast cancer cell line MDA MB 231 that express hyaluronan receptor (CD44), as
shown in FIG 13 and Table 1.
Page 16 of 21 LI1011-MSMF-001-PR- Provisional
[094] Table 1. IC 50 (nM) values of MDA-MB-231, HaCaT, HEK293T and NIH 3T3 after
treatment with different nanoformulations (Drug: Paclitaxel (control), HC-CN+Drug,
HA+CN NPs and HA NPs) after 48 hr incubation. (Drug: Paclitaxel; CN: chitosan, HA:
Hyaluronic acid)
[095] In vitro cytotoxicity study by MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-
Diphenyltetrazolium Bromide] assay (Chitosan-HA-Dox NPs)
[096] Breast cancer cell line MDA MB 231 and normal cell lines HaCaT, nih3T3 were
treated with doxorubicin carrying nanoparticles with respective controls for both 48 hours
and 72 hours.
[097] In case of free doxorubicin treatment, the IC50 values are significantly higher in
MDA MB 231 cells in comparison to HaCaT and NIH 3T3 cells (48 hr and 72 hr). But In
case of HC-Dox treatment the IC50 values are significantly lower in MDA MB 231 cells
in comparison to HaCaT and NIH 3T3 cells (48 hr and 72 hr). So, nanoparticles loaded
with doxorubicin effectively killed breast cancer cell line MDA MB 231 and in
comparison to normal cell lines HaCaT, NIH 3T3. In MDA-MB-231 cells, the IC50 values
(nM) of HC Dox are significantly lower than the CN Dox (p<0.001) and HA Dox (p<0.05)
after 48 hr incubation. Similarly, the IC50 values (nM) of HC Dox are significantly lower
than the CN Dox (p<0.001) and HA Dox (p<0.01) after 72 hr incubation. Also in MDAMB
231, the IC 50 values of HA-Dox are significantly lower than CN-Dox (both after
48hr and 72 hr) that might be due to the higher internalization of HA-Dox in comparison
to CN-Dox due to HA specificity to cancer cells (MDA MB-231), as shown in FIG 14,
FIG 15 and Table 2
[098] .
Page 17 of 21 LI1011-MSMF-001-PR- Provisional
[099] Table 2. IC 50 (nM) values of MDA-MB-231, HaCaT, nih 3T3 after treatment
with different nanoformulations (Dox: Doxorubicin (control), HC Dox: Chitosan-
HA-Doxorubicin NPs, CN Dox: Chitosan-Doxorubicin NPs, HA-Dox: HA
Doxorubicin NPs) after 48 hr and 72 hr incubation.
[0100] Merely for illustration, only representative number/type of graph, chart, block and
sub- block diagrams were shown. Many environments often contain many more block and
sub- block diagrams or systems and sub-systems, both in number and type, depending on
the purpose for which the environment is designed.
[0101] Important embodiments of Chitosan-HA-Paclitaxel nano particulate system
1. Successful conjugation of chitosan nanoparticles with HA by carbodiimide reaction with
EDC and NHS as catalysis that effectively controls higher encapsulation of drug Paclitaxel
(Encapsulation efficiency of drug: 80.8%).
2. The sustainable and specific pH responsive release of Paclitaxel was observed from
Chitosan-HA-Paclitaxel NPs at pH 5.2 (48.26% at 24 hr, 64.24% at 48 hr, 70.67% at 72
hr).
3. The HA (ligands for the receptor CD44) was exposed to the surface of the nanocomplex
(revealed from zeta potential analysis, FTIR analysis study and relative composition
analysis).
4. In vitro assay (cell culture study) confirmed the specificity of this system that avoids
systemic toxicity or very minimal side effects, as Chitosan-HA-Paclitaxel nanoparticles
selectively killed only breast cancer cell line MDA MB 231 and did not affect the normal
cell lines HaCaT, HGF, and NIH3T3.
[0102] According to a non limiting exemplary aspect of the present invention, the
nanoparticles are designed in a way that they will specifically target the cancer cells.
Encapsulating drug into the nanoparticle increases systemic life span of the drug and nonspecific
cells are not affected, in addition there is no systemic toxicity or very minimal
side effects.
[0103] According to a non limiting exemplary aspect of the present invention, the tumor
heterogeneity is responsible for inefficiency of chemotherapy or tumor relapse, for this
reason multiple drugs can be encapsulated in the aforementioned nanoparticulate, which
can be used as the combinatorial chemotherapy.
[0104] Yet another aspect of the present invention, this nanoparticulate system can be used as
novel delivery system for multiple drugs to attain combined therapeutic index.
Page 18 of 21 LI1011-MSMF-001-PR- Provisional
[0105] According to a non limiting exemplary aspect of the present invention, this
nanoparticulate system can be made widely useful by using different receptors/ligands
specific to different types cancer.
[0106] Yet another aspect of the present invention, chitosan/ligands can be functionalized
with different chemical groups to make this system more specific and effective.
[0107] According to a non limiting exemplary aspect of the present invention, one of the
mode to practice the aforementioned invention could be intravenous injection of
nanoparticle suspension to patients.
[0108] While specific embodiments of the invention have been shown and described in detail
to illustrate the inventive principles, it will be understood that the invention may be
embodied otherwise without departing from such principles.
[0109] Reference throughout this specification to “one embodiment”, “an embodiment”, or
similar language means that a particular feature, structure, or characteristic described in
connection with the embodiment is included in at least one embodiment of the present
invention. Thus, appearances of the phrases “in one embodiment”, “in an embodiment”
and similar language throughout this specification may, but do not necessarily, all refer to
the same embodiment.
[0110] It should be understood that the figures and/or screen shots illustrated in the
attachments highlighting the functionality and advantages of the present invention are
presented for example purposes only. The present invention is sufficiently flexible and
configurable, such that it may be utilized in ways other than that shown in the
accompanying figures.
References
1) P. Calvo, C. Remu˜nán-López, J.L. Vila-Jato, M.J. Alonso, Novel hydrophilic chitosan–polyethylene oxide
nanoparticles as protein carriers, Journal of Applied Polymer Science 63 (1997) 125–132.
2) Marcel J.E. Fischer. Amine Coupling Through EDC/NHS: A Practical Approach. N.J. de Mol, M.J.E. Fischer
(eds.), Surface Plasmon Resonance, Methods in Molecular Biology 627, DOI 10.1007/978-1-60761-670-2_3.
Page 19 of 21 LI1011-MSMF-001-PR- Provisional
CLAIMS
I Claim,
1) A nanoparticle delivery system, comprising: a self-assembling reconstituted high
density lipoprotein complex comprising a combination of:
a) a pH-specific polymer;
b) a cross linker;
c) a receptor binding component (R); and
d) a drug (D)
wherein the pH-specific polymer crosslinked to receptor binding component
(R) and Drug (D) encapsulated within the nano matrix, and the selfassembling
reconstituted high density lipoprotein complex has a diameter in
the range of from about 70 nm to 110 nm.
2) The nanoparticle delivery system of claim 1, wherein the pH-specific polymer
comprises a chitosan component.
3) The nanoparticle delivery system of claim 1, wherein the system is a specific
combination of a cancer cell-specific receptor and a particular ligand, wherein specific
receptors/ligands are functionalized with specific chemical groups to make this
system specific and effective for a specific cancer type.
4) The nanoparticle delivery system of claim 1, wherein the nanoparticle delivery
vehicle further comprises a pharmaceutical agent.
5) The nanoparticle delivery system of claim 6, wherein the pharmaceutical agent is
located in the core of the nanoparticle delivery vehicle, wherein the core of the
nanoparticle represents a lipophilic and nonaqueous environment.
6) The nanoparticle delivery system of claim 6, wherein the pharmaceutical agent is a
lipophilic or hydrophilic.
7) The nanoparticle delivery system of claim 1, wherein the lipid binding protein
component is modified to enhance the targeting efficacy of the drug.
8) The nanoparticle delivery system of claim 1, wherein the lipid binding protein
component is modified by the attachment of antibodies, folic acid residues or other
ligands that target the surface of malignant cells and tumors.

9) The nanoparticle delivery system of claim 1, further comprising a functional moiety which augments the efficacy of the pharmaceutical agent through interaction with a cell surface receptor.
10) A method for delivering a drug of interest to a subject, comprising: administering the nanoparticle delivery system of claim 6 to the subject; wherein the pharmaceutical agent is the drug of interest for cancer.
11) The method of claim 10, wherein the nanoparticle delivery system is delivered intravenously, subcutaneously, parenterally, intramuscularly, transdermally or transmucosally.
,CLAIMS:I Claim,

1) A nanoparticle delivery system, comprising: a self-assembling reconstituted high density lipoprotein complex comprising a combination of:
a) a pH-specific polymer;
b) a cross linker;
c) a receptor binding component (R); and
d) a drug (D)
wherein the pH-specific polymer crosslinked to receptor binding component (R) and Drug (D) encapsulated within the nano matrix, and the self-assembling reconstituted high density lipoprotein complex has a diameter in the range of from about 70 nm to 110 nm.
2) The nanoparticle delivery system of claim 1, wherein the pH-specific polymer comprises a chitosan component.
3) The nanoparticle delivery system of claim 1, wherein the system is a specific combination of a cancer cell-specific receptor and a particular ligand, wherein specific receptors/ligands are functionalized with specific chemical groups to make this system specific and effective for a specific cancer type.
4) The nanoparticle delivery system of claim 1, wherein the nanoparticle delivery vehicle further comprises a pharmaceutical agent.
5) The nanoparticle delivery system of claim 6, wherein the pharmaceutical agent is located in the core of the nanoparticle delivery vehicle, wherein the core of the nanoparticle represents a lipophilic and nonaqueous environment.
6) The nanoparticle delivery system of claim 6, wherein the pharmaceutical agent is a lipophilic or hydrophilic.
7) The nanoparticle delivery system of claim 1, wherein the lipid binding protein component is modified to enhance the targeting efficacy of the drug.
8) The nanoparticle delivery system of claim 1, wherein the lipid binding protein component is modified by the attachment of antibodies, folic acid residues or other ligands that target the surface of malignant cells and tumors.
9) The nanoparticle delivery system of claim 1, further comprising a functional moiety which augments the efficacy of the pharmaceutical agent through interaction with a cell surface receptor.
10) A method for delivering a drug of interest to a subject, comprising: administering the nanoparticle delivery system of claim 6 to the subject; wherein the pharmaceutical agent is the drug of interest for cancer.
11) The method of claim 10, wherein the nanoparticle delivery system is delivered intravenously, subcutaneously, parenterally, intramuscularly, transdermally or transmucosally.