FIELD OF INVENTION

The present invention is related to core-shell photo-chemo nanomedicine intended for combinatorial delivery of a photosensitizer drug and chemo drug against disease like cancer. More particularly, the present invention provides a method for synthesizing stable, well dispersed, unagglomerated core-shell nanoparticles of total size <1000nm, loaded with a photosensitizer drug and chemo-drug at least one each in the core and shell. More specifically, the invention is related to i) polymer-core/protein-shell and ii) polymer-core/polymer-shell nanoparticles, each core or shell are loaded with at least one photosensitizer drug or chemo-drug, separately.

These nanomedicines are used for the treatment of cancer type diseases using combinatorial photodynamic therapy (PDT) and chemotherapy.

BACKGROUND

Cancer, one of world's deadliest diseases and leading cause of death, is operated by several altered molecular pathways. Regardless of the improvements in diagnostic tools and sophisticated treatment approaches, it's highly heterogeneous genotypes and patient to patient variations make the cure very difficult.

Conventional therapies such as chemotherapy and radiation therapy mainly focus on killing cancer cells by causing DNA damage by direct ionization, intercalation or reactive oxygen stress. New molecular targeted therapies cause inhibition of aberrant protein kinase or enzymatic activity that supports cancer survival. However, none of these methods could provide significant curative effect for cancer.

One of the main reasons is that cancer is operated by multiple molecular mechanisms involving complex protein kinase networks, DNA replication, mutations, drug resistance, and existence of cancer stem cells having privileged advantages of enhanced ROS scavenging capacity and preferentially activated DNA repair mechanism.

Photodynamic therapy (PDT) is a promising method of treating cancer type disease using photosensitive chemicals that release reactive oxygen species upon excitation with light, in the presence of oxygen. This highly reactive ROS species kill malignant cells by apoptosis and/or necrosis along with shut down of tumour microvasculature and stimulation of the host immune system. The neoplastic conditions mainly treated by PDT includes inoperable esophageal tumors, head and neck cancers, micro-invasive endo-bronchial non-small cell lung carcinoma, and cancers in bile duct, gall bladder, gullet, lung, mouth, skin etc. PDT is also being investigated in preclinical and clinical studies for breast, prostate and ovarian cancers.

Even though PDT possess high potential for cancer treatment, the effectiveness of this therapy is limited by the problems associated with non-specific light toxicity, delivery of PS drugs, and the ability of cancer stem cells to survive PDT by scavenging ROS very effectively.

ROS, being is the main mediator of PDT assisted cancer cell death, its inhibition by cancer stem cells due to its enhanced ROS scavenging ability and DNA repair capacity pose a major challenge for PDT to cure cancer completely. Abrogation of such drug-resistant mechanisms could have major therapeutic implications in PDT.

One of the possible methods to enhance the therapeutic efficacy of PDT is to inhibit the molecular mechanism responsible for ROS resistance or DNA repair mechanisms of cancer cells using other chemical drugs such as small molecule inhibitors that can down regulate the respective intracellular pathways. For example, simultaneous use of small molecule drugs that inhibits the ROS scavenging activity in cancer along with photosensitizes can improve the effectiveness of PDT. Alternatively, inhibition of DNA repair process using small molecule drugs followed by application of ROS stress by PDT, or inhibition of metastasis / migratory capacity of cancer followed by application of ROS stress by PD, etc, are promising combinatorial approach to achieve better curative effects.

In addition, studies have reported the important role of cancer micro environment in attaining drug resistant and uncontrolled proliferation of cancer. It is understood that destroying cancer micro environment is critical in achieving better therapeutic effects. One possibility is to use molecules that disrupt the cytoskeletal arrangement and cancer cell-niche interactions by inhibiting the integrin binding capacity, in combination with application of ROS stress by PDT may yield better curative effects.

Conceivably, combinations of agents with complementary mechanisms of action could be more effective in dealing with cancer than single agents or single therapy.

In another aspect, cancer cells in advanced stages of the disease frequently exhibit multiple genetic alterations leading to ineffectiveness of conventional single agent chemotherapy. Development of drug resistance is another major obstacle to the success of anticancer chemotherapy. Tumor cells utilize multiple mechanisms to reduce the accumulation of the anticancer agents at its intracellular site of action or develop pint mutations in the drug binding domain of the kinase. In order to overcome these challenges, it is proposed to apply multiple stress mechanisms against cancer simultaneously. For example, application of DNA intercalating agents together with ROS stress by photodynamic therapy, or silencing of oncogene using small interfering RNA together with application of PDT , etc are promising options.

However, all the above methods demands simultaneous delivery of chemical drugs together with photosensitive drugs to cancer. The success of such combinatorial approaches lies heavily of our ability to deliver appropriate drug combinations at appropriate concentrations, specifically at the tumour sites. Overcoming the challenges faced by such a strategy includes attaining optimum drug concentrations of drugs at the tumour sites in appropriate sequence with least systemic toxicities.

The emerging field of nano-drug delivery offer great promise for targeted delivery of drugs to tumor type diseases. Nanoparticle mediated drug delivery improves the conventional cancer therapy by aiding the delivering of appropriate drug combinations in optimum therapeutic dosages on the tumour site and also by minimising the risk systemic toxicities. They also aids targeted drug delivery by conjugating drug loaded nanoparticles with tumour specific bio-markers like antibodies, peptide and other ligands.

Although there are number of nanoparticle based drug delivery systems reported so far, most of them deal with single agent delivery. Simple nanoparticle aided drug delivery systems possess architectural limitations to carry multiple therapeutic agents in optimum concentrations and also to give the desired drug release. There are also risks regarding possible cross-talk between the drugs and photo-degradation of chemodrugs by the photosensizers. So a heterogeneous drug delivery system like core-shell nanoparticles were two drug molecules can be incorporated in each layer without cross-interactions are needed. The core and shell can be made up of different materials such as polymers or proteins according to the nature of the drug, its sequence of delivery and the release kinetics needed. In such a platform a conventional cytotoxic stress inducing agent like PS or DNA damaging agents or other ROS agents can be used along with its drug resistance inhibitors like DNA damage inhibitors (PARP inhibitor, Chkl& Chk2 inhibitors and like), ROS scavenging inhibitors, cytoskeletal disturbing agents (for example dasatinib) etc.

PRIOR ART

Biodegradable or biocompatible polymeric and protein nanoparticle were reported to be used for delivering single or multiple therapeutics including chemo-drugs, pro-drugs, contrast agents for treating and diagnosing various diseases and disorders; especially cancer and related manifestations. Majority of such nanoparticle mediated delivery of therapeutics also possess provision for attaching a specific ligand to aid the targeted drug delivery. In prior art use of nanoparticles with and without a targeting ligand for the delivery of chemotherapeutic or imaging agent is described in patents US 006/165440, US 20080181852, US 20090226393, US 20110020457, US 2007/0009441, US 2008/0253969, etc. Use of polymeric nanoparticles for photosensitizer delivery is deliberated in patents US 2011/10238001, US 2011/0022129 etc. US 20110165258 disclosed the use of polymer aggregates having both hydrophilic and hydrophobic segments for the delivery of drugs and diagnostic agents.

Simultaneous loading of pro-drug along with its activating enzyme in nanoparticles for two step targeted tumour therapy has disclosed in US patent 2011/10217363.

Polymeric micelle having a structure of core and shell were reported to be used for drug delivery applications, were disclosed in patents EP 0552802, US 6080396, US 5449513 etc. US 201110091534, US 2010/0159019, US 7638558 disclosed multi-block polymeric micelles for drug delivery applications. US 8021652 disclose the preparation and use of biodegradable branched polylactide derivatives forming polymeric micelles for delivery of poorly water soluble drugs. US patents 2011/10027172 and 201 l/10229556discloses use of lipid coated polymeric particles for drugs & radiopharmaceutical agents and adjuvant molecules respectively. Amphiphilic block co polymer and polymeric composition comprising the same for drug delivery applications are disclosed in US 7311901 & US 2008/0152616. In the prior art, the preparation of nanoparticles with micellar structure comprising an amphiphilic block copolymer with a hydrophobic agent encapsulated within the micellar structure, and a functionalized corona (US patent no: 2007/0253899), drug-loaded micelle comprising a triblock copolymer, wherein said micelle has a drug-loaded inner core, a crosslinked outer core, and a hydrophilic shell, wherein the multiblock copolymer (US patent no: 2010/0159020), drug-loaded poly(alkyl-cyanoacrylate) nanoparticles (US patent no: 2008/0182776) etc are reported.

Conversely patent US 2009/0214633 discloses the use of particles prepared by nano-encapsulation having lipid core and polymeric shell for protein drug delivery applications. US 2008/0248126 discloses use of multi-layered polymeric or oligomeric nanoparticles prepared by ring opening polymerisation for drug delivery applications, in that the drug is either loaded in the core or in shell. In a similar art disclosed in US patent 2010/0203149, nanoparticle having drug loaded polymer core surrounded by water soluble polymeric chains for its potential use in cytoplasmic drug delivery to cancer cells.

Use of polycaprolactone (PCL) or Polyethylene glycol (PEG) coated polymeric nanoparticles for anticancer drug delivery was disclosed in US patent 2011/0052709. In this, the drug is loaded in the polymer core made of cellulose or collagen or lactose or alginate by milling process. The outer polymer coat is not carrying any therapeutic or imaging agents. Polymer dendrimers having a core-shell nature were disclosed for their use in delivering multiple therapeutic agents, photosensitizers, contrast agents or biologically active agents (DNA, RNA etc.,) in US 2005/0281777 & US 2009/0012033
Even though nanoparticle having a core-shell nature or structure like multi-layered dendrimers, polymeric micelles and polymer or lipid coated nanoparticles were disclosed in the above mentioned prior arts, none of these arts have complied with a definitive structure of a nano-construct comprising of a distinct polymeric core and a distinct polymer /protein shell loaded with a photosensitizer and chemo-drug in combination to aid a combinatorial treatment using PDT and chemotherapy togother. Contrary to the earlier inventions, we have synthesized a polymer-core and polymer/protein shell nanomedicine, each having a cross linked/solid structure with a distinct interphase.

Even though the polymeric nanopartices were used in few of the prior arts, polymer-protein core shell systems are not reported. Furthermore, in our method, these protein molecules are cross-linked for better encapsulation of drug molecule. The cross-linking methods are selected in such a way that the chemical stability of drug payloads remains unchanged. Accordingly, there exist no prior art on the preparation of polymer-core/protein shell or polymer-core/polvmer-shell nanomedicine encapsulating photosensitizer and chemo drugs separately or combined in core-shell nanoparticle aiding the sequential or simultaneous delivery of these drugs at the site of action, in a targeted manner.

SUMMARY OF THE INVENTION

In the present invention, a multifunctional polymer/protein and polymer/polymer core-shell photo-chemo nanomedicine that can provide simultaneous or sequential delivery of two different types of therapeutic agents such as a photosensitizer drug in combination with an anti-neoplastic agent to cancer and related diseases in a targeted fashion. Specifically, the nanomedicine is based on a polymeric nano-core and a shell made up of either another polymer or protein. The core and the shell will be loaded with drugs in the following possible formats: a) core with a photosensitizer and shell with anti-neoplastic chemodrugs, b) core with antineoplastic chemodrugs and shell with a photosensitizer.

The core and shell are made up of bio-compatible polymers or proteins suited for the dug loading and preferred controlled release. This unique nanomedicine is intended for the combinatorial cancer treatment using photodynamic therapy and chemotherapy.

DEFENITIONS

The term "nanoparticle" as used herein refers to primary inventive nanoparticles formed by protein or polymer, measuring size about 1-1000 nm, preferably l-100nm, most preferably around 1-50 nm in size showing "multifunctional' property of delivering multiple therapeutic agents such as photosensitizer, chemo-drugs, small molecule inhibitors etc., in different combinations of at least one photosensitizer and one or more chemo-drugs together.

The term "core-shell nanoparticle" as used herein refers to a heterogeneous nanoconstruct formed with a central nanoparticle core and an outer nano-shell and both have a distinct interphase.

The term "therapeutics" as used herein refers to photosensitizers, chemo-drugs, small molecule inhibitors, pro-drugs, etc that have a therapeutic effect against a diseases, especially cancer and related clinical manifestationss.

The term "targeting ligand" as used herein refers to biomolecules that can specifically identify and target another molecule like an antigen or receptor on the surface of cell-membrane of diseased cells such as that of cancer / tumor. Targeting ligand include antibodies, peptides, aptamers, vitamins like folic acid, sugar molecules like mannose ligands, carbohydrates etc.

The term "nanomedicine' as used herein refers to nanoparticles loaded with therapeutics.

The term "photo-chemo nanomedicine' as used herein refers to a nanoparticle loaded with at least one photosensitizer and chemo-drug in a single core-shell nanomedicine system.

FIGURE CAPTIONS:

Figure 1: Schematic showing preparation of polymer-protein core-shell nanomedicine construct made of PLGA-mTHPC core & albumin-dasatinib shell

Figure 2: A) Atomic force microscopic image and B) DLS analysis of PLGA-mTHPC nanocore showing particle size of ~ 80nm. C) SEM image of PLGA-mTHPC:BSA-dasatinib core-shell nanomedicine, Inset is TEM image of core-shell nanomedicine showing the difference in contrast between the two D) DLS analysis of core-shell nanomedicine showing an overall size of ~120nm,

Figure 3: FTIR spectrum of PLGA-mTHPC core, albumin-dasatinib shell and the complete nanomedicine construct.

Figure 4: Figure A represents the comparison of florescence property of bare mTHPC and nano-core encapsulated mTHPC. B) is the singlet oxygen generation by the nano-core by SOSGR assay .

Figure 5: Cellular uptake studies of nanomedicine construct by U87MG human glioma cells. A) Dot plot showing nanomedicine uptake determined FACS analysis. B) Concentration and time dependent pattern of nanomedicine uptake exhibited by U87MG cells C) Florescence image showing mTHPC florescence from nano-core inside the cells after 6hr incubation

Figure 6: In vitro cell scratch assay for migration inhibition analysis. A) Microscopic images of in vitro scratch assay showing effective inhibition of cell migration on the nanomedicine treated cells. B) and C) are the quantitative representation of migration inhibition in terms of no of cell migrated and percentage scratch healed respectively. D is western blot analysis showing p-Src down regulation by the nano-shell encapsulated dasatinib.

Figure 7: Confocal microscopic images showing actin cytoskeleton disruption in the core-shell nanomedicine treated glioma cells

Figure 8: Effect of nanomedicine treatment on the morphology and attachment ability of U87MG cells. Figure A is SEM images showing significant reduction in philopodial extensions in the treated cells in comparison with normal cells. B is confocal imaging of actin (red) and vinculin (green) showing reduction in focal adhesion point formation by nanomedicine treatment.

Figure 9: Confocal-DIC imaging of ROS generation; DCFHDA florescence (green) corresponds to extend of ROS generation.

Figure 10: Analysis of cytotoxicity by the nanomedicine construct. Graph A showing absence of dark toxicity of nanomedicine. Figure B showing light induced cytotoxicity by the nanomedicine construct and nano-core. Figure C is early stage apoptosis induced by nanomedicine mediated combination therapy.

DETAILED DESCRIPTIONS OF THE INVENTIONS

The main feature of the inventive nanomedicine is the core-shell structure where the core is formed by a biocompatible and biodegradable polymer loaded with either a photosensitizer drug or a chemo-drug and the shell formed over the core, either by another polymer or a protein and loaded with either a photosensitizer or a chemo-drug.

As the above nanomedicine is intended to combine photodynamic therapy with chemotherapy, the core-shell structure will always have a photosensitizer and chemo-drugs in combination, for example, in case of a metastatic cancer, aberrant kinase associated with migration of cancer cells can be inhibited by a chemo-drug loaded in the shell and photosenzitizer drug in the core. This facilitate application of PDT assisted ROS stress after curtailing the migratory capacity of metastaic cells. An advantage of this method is that, molecular pathways responsible for migration and metastasis can be inhibited by relatively less concentration of small-molecules and complete cell death can be achieved by PDT.

In another embodiment, enhanced PDT mediated cytotoxicity can be achieved by delivering an antioxidant inhibitor molecules along with photo-sensitizer using a core-shell nanomedicine. It is well known that radiation resistant cancer cells have evolved various cellular anti-oxidant pathways to counteract radiation/PDT or chemotherapy mediated ROS stress. In addition, a small populations of cancer stem cells, are the main causes of cancer relapse and drug resistance as they exhibit elevated activity of such antioxidant pathways and over expression of ROS scavengers. A combinatorial cancer treatment using a photo-drug along with antioxidant inhibitors like diethyl-dithiocarbamate, methoxyestradiol, 1-buthionine sulfoximine, 3-amino-l,2,4-triazole, etc may potentiate the PDT mediated cytotoxicity.

In yet another embodiment, another radiation or chemo resistance mechanism of cancer or cancer stem cells are preferential activation of DNA repair mechanism. In such case, a small molecule inhibitor of enzymes responsible for activated DNA repair damage can be loaded on to the shell of the core-shell nanomedicine together with a photomedicine at the core. In such case, the DNA damage caused by ROS due to PDT cannot be effectively repaired by the cancer cells, thus achieving better toxicity effects even inn radiation resistance or cancer stem cells. Small molecule inhibitors for such DNA damage repair molecules include PARP inhibitors, Chk inhibitors, etc.

In a preferred embodiment of the said core-shell nanomedicine, the polymer for making nano-core and nano-shell is selected from the group of biodegradable polymers such as, but not limited to poly glycolic acid (PGA), poly(lactic-co-glycolic acid) (PLGA), glycolide/trimethylene carbonate copolymers (PGA/TMC); poly-lactides (PLA), poly-L-lactide (PLLA), Poly-DL-Iactide (PDLLA), L-lactide/DL-lactide copolymers; lactide/tetramethyl-glycolide copolymers, poly-caprolactone (PCL), poly-valerolacton(PVL), poly-hydroxy butyrate (PHB), poly vinyl alcohol (PVA) poly-hydroxy valerate(PHV), polyvinylpyrrolidone (PVP), Polyethyleneimine (PEI) and lactide/trimethylene carbonate copolymers, chitosan, carboxymethyl chitosan, chitin, pollulan, etc., or blends thereof.

In a preferred embodiment of the said core-shell nanomedicine, the protein used for nano-shell is chosen from the group of protein such as human serum albumin, or protamine, transferrin, lactoferrin, fibrinogen, gelatin, mucin, soy protein, apoferritin, ferritin, lectin, lactoferrin, gluten, whey protein, prolamines such as gliadin, hordein, secalin, zein, avenin, or combinations thereof.

In a preferred embodiment of said core-shell nanomedicine, the photozensitizer loaded on either nanomediciene core or shell is chosen from, and not limited by chlorine e6 (Ce6), meso-tetra(3-hydroxyphenyl)chlorin (m-THPC), methylene blue, benzoporphyrin derivative monoacid ring A (BPD or verteporfin), photofrin, Rose bengal, metal phthalocyanine, hypericin, toluidine blue O, pyropheophorbide-a hexyl ether (HPPH), Indium pyropheophorbide, padoporfin, padeliporfin, and combinations thereof.

In a preferred embodiment of the said core-shell nanomedicine, the chemo-drug loaded on either nanomediciene core or shell is chosen from, but not limited by anti-neoplastic agentssuch as cyclophosphamide, methotrexate, 5-fluorouracil, doxorubicin, cisplatin, hydroxyurea, leucovorin calcium, tamoxifen, flutamide, asparaginase, altretamine, mitotane, procarbazine hydrochloride, mechlorethamine, thioguanine, carmustine, lomustine, temozolomide, melphalan, chlorambucil, streptozocin, methotrexate, vincristine, bleomycin, vinblastine, vindesine, dactinomycin, daunorubicin, doxorubicin, tamoxifen or anti-proliferative agents such as rapamycin, paclitaxel, or anti-angiogenesis agents such as avastin, or inhibitors of tyrosine kinase including epidermal growth factor receptor (EGFR), vascular endothelial growth factor receptor (VEGFR), platelet derived growth factor receptor (PDGFR), fibroblast growth factor receptor (FGFR), Rous sarcoma oncogene/Breakpoint cluster region/Abl (Src-bcr-abl), Insulin-like growth factor 1 receptor (IGF-1R), FLT-3, HER-2, STAT5, c-Kit, c-Met, ALK, ETA receptor inhibitor, HIF inhibitor, Syk inhibitor, Tie2 kinase inhibitor and the like), Vascular disrupting agents (e.g. plinabulin), cell cycle/check point inhibitors like polo-like kinase (PLK) inhibitor (e.g. volasertib), cyclin dependent kinase (CDK) inhibitors (e.g. seliciclib, indirubin etc.,), topoisomerase inhibitors (e.g. adriamycin, camptothecin, etoposide, idarubicin, irinotecan, topotecan, mitoxantrone etc.,), microtubule inhibitors (e.g. docetaxel, paclitaxel, vincristine etc.,), antimetabolites (e.g. decitabine, gemcitabine, fludarabine etc.,), telomerase inhibitors, DNA & RNA replication inhibitors (e.g. clarithromycin, cytarabine, mitoxantrone HC1 etc.,) dihydrofolate reductase inhibitor, HDAC inhibitor, Bcl-2 and TNF-a inhibitors, PARP inhibitors, MAPK inhibitors, PI3K/Akt/mTOR inhibitors, integrase and protease inhibitors, Wnt/Hedgehog/Notch inhibitors, cAMP, lipide signaling inhibitors (e.g. PKC, PIM etc.,), TGF-P inhibitors, chemotherapeutic pro-drugs like and the combinations thereof.

In a preferred embodiment of said core-shell nanomedicine, the polymer core or shell is formed by a proffered method chosen from spontaneous emulsification, solvent diffusion, salting out, emulsification-diffusion method, supercritical fluid technology, micro emulsion or double emulsion, electrospray, electro deposition, ultrasonication, spray drying etc.

In a preferred embodiment of said core-shell nanomedicine, the protein shell is formed by a proffered method chosen from simple desolvation or co-acervation, complex co-acervation, nano-precipitation, sol-gel, spray drying slating-out or cross linking.

In a preferred embodiment of said method of making core-shell nanomedicine, the nanomedicine-core and shell have clear separating between each other having distinct interphase between the core and shell. This designed architecture prevent possible crosstalk and adverse interactions between the different therapeutics loaded in each part

Referring to the schematic given in Fig. 1, for the preparation of polymer-core/protein-shell photo-chemo nanomedicine, in step-1, a drug loaded polymer core is formed by first treating the desired drug-1 (for example a photosensitizer, mTHPC) with its suitable carrier polymer (for example PLGA) and precipitated as nanoparticles using suitable method, for example electrospray or micro-emulsion.

The precipitated particles are purified by washing and dried by lyophilization. In the next step-2, a suitable protein, for example albumin, is treated with the desired drug-2, for example a chemodrug, Dasatinib, for electrostatic binding. In the third step, the lyophilized polymer core nanoparticles are suspended in the protein-drug-2 mixture and the method of coaservation is applied to form the shell. In the step 4, the core-shell nanoparticle is bio-conjugated with specific ligands for example folic acid, aid targeted delivery.

In relation to the above method of preparing embodiment, the nanomeidicine core showed an average size of-80 nm and an overall size of ~120 nm as shown in the figure 2. Figure 2A and 2B denotes the AFM image and DLS measurement of ~80nm sized PLGA-mTHPC core, where 2C and 2D denotes the SEM image and DLS measurements of ~120nm sized nanomedicne construct. The contrast difference between the nano core and shell clearly visualized in the TEM image (Fig 2C inset) establishes our claim of core-shell nanomedicine. The nanomedicine construct showed an overall size of ~ 120 nm as depicted in figure.

In yet another aspect of the above mentioned embodiment, the nanomedicne core, shell and the entire nanomedicine construct shows distinct FTIR pattern as depicted in figure 3, shows the successful incorporation of the specific therapeutics in the nanomedicine construct. The IR peaks at 3430cm-land 1647cm-1 for PLGA-mTHPC possibly represent hydroxyl groups and primary/secondary amine groups of mTHPC respectively. Presence of aromatic amines in the porphyrin structure of mTHPC was evident from IR peak at 1288cm-1. This peak may also represent the presence of carboxylic and ester linkages in the PLGA matrix. Also, the presence of ketone groups in the PLGA matrix was confirmed by IR peak at 1121cm-1. All these indicated that mTHPC restores its native intact structural chemistry even after encapsulation within PLGA, despite electrospray invokes high voltage to produce PLGA-mTHPC nanoparticles. IR peaks of core-shell nanomedicine showed additive IR peaks of PLGA-mTHPC and BSA-dasatinib.

In yet another aspect of the above embodiment, the nanomedicne core show enhanced flurescence when compared with the same concentrations of photosensitizer at lkmax 652nm in aqueous medium. The figure 4A shows the 38 fold increase in florescence intensity by the PLGA-mTHPC nano-core in comparison with same concentrations of bare photosensitizer. Figure 4B depicts the singlet oxygen generation from the nano-core under laser irradiation measured by SOSGR assay, providing proof of its capability to induce enormous ROS stress to the diseased cells. PLGA-mTHPC released singlet oxygen at least 25 times greater than free mTHPC of equimolar concentration in aqueous medium. A steady state increase in the release of singlet oxygen from PLGA-mTHPC for 50 minutes was found when singlet oxygen is detected at emission λmax 525nm during light irradiation.

In yet another aspect of the same embodiment, the PLGA-mTHPC/albumin-Dasatinib nanomedicine shows a time and concentration dependent cell uptake pattern. Fig. 5A refers to the uptake studies of the nanomedicine by FACS analyzing, showing an effective particle uptake by U87MG glioma cells after 6hr incubation. Refrering to fig. 5B depicts a quantitative data showing time and concentration dependent pattern of nanomedicine uptake as a measure of mTHPC florescence. The mTHPC florescence from the nanomedicine treated cells, visualized under florescent microscope, depicted in figure 5C.

In yet another aspect of the above embodiment, the cell migration inhibition properties of the core-shell nanomedicine is depicted in the figure 6. Fig 6A depicts the microscopic images of effective inhibition of cell migration.. The nanomedicine treated cell showed effective inhibition in migration even after 18 hrs. while the untreated cells migrated and healed the scratch area. The quantitative measure of the same is depicted in figure 6B and 6C by means of total no. of cells migrated and the percentage scratch healing respectively. The p-Src level down regulation by nanomedicine-dasatinib, that intern inhibit cell migration is proved by western blot analysis depicted in fig. 6D. The nanomedicine treatment induced changes in cytoskeleton arrangement investigated is depicted in figure 7. The actin staining-confocal images of U87MG cell showed loss of proper cytoskeleton in the drug treated group. Loss of philopodial extension (fig 8A) and focal adhesion points (8B) were also seen in the nanomedicine treated cells.

In yet another aspect of the above embodiment, the nanomedicine induced intracellular ROS was visualized by DCFH staining and live cell DIC imaging. The nanomedicine treated cells showed enhanced production of intracellular ROS compared to bare photosensitizer and showed morphological changes associated with apoptosis (fig. 8). The photo-chemo nanomedicine treated cells showed enhanced cytotoxicity compared to cells treated with nanoparticles having photodrug alone (fig 9). The dasatinib in the nano shell enhanced the PDT mediated cytotoxicity. The confocal images of the cells, one hour past the light irradiation (fig. 9B) shows the early stage apoptosis; Annexin V (FITC conjugated) bound to the externalized phosphatidyl serine as a result of apoptosis.

The authors have invented a core-shell nanomedicine having a clear interface for delivering a photosensitizer drug and a chemo-drug simultaneously/sequentially for combinatorial cancer treatment to aid enhanced cytotoxicity to cancer cells. The design of the nanomedicine is in such a way to simultaneously carry two different drugs and deliver it specifically to the tumor cells in desired fashion. The targeting is achieved by a specific biomarker ligand conjugated to the nanomedicine construct. The core and shell are made by different polymers or a protein and polymer aiding the optimum release kinetics.

Examples

Example 1: Polymer core/protein shell photo-chemo-nanomedicine.

(PLGA-mTHPC core/Albumin-Dasatinib shell)
In this example preparation of a combinatorial polymer-protein core-shell nanomedicine with photsensitizer meta-tetra (hydroxyphenyl) chlorine (mTHPC) loaded polymeric [PLGA: Poly(lactic-co-glycolic acid (50:50)] nanocore and small molecule inhibitor dasatinib entrapped protein shell is presented. PLGA solution containing ImM mTHPC was prepared by dissolving it in 1.5 wt % PLGA solution in acetone and was stirred for 1 hr in dark prior to electrospray. This solution was then electrosptrayed at a rate of 1.5 ml/hr to a grounded glass beaker containing de-ionized water premixed with 0.001v/v Tweeen 20® under constant stirring (550 - 600 rpm). The tip to target distance was maintained as 7.5 throughout the experiment. The electrospray was carried out under ambient temperature, pressure and 55±5% humidity, by applying a potential of 1.4kV/cm using a high voltage supply.

The particles were acquired by lyophilizing the electrosprayed solution. The Albumin-dasatinib solution was prepared by mixing fresh dasatinib-DMSO stock in 5-10 wt % BSA solution in de-ionized water to get a final dasatinib concentration of 10 uM, Lyophilized PLGA-mTHPC particles were mixed with the BSA-dasatinib solution and stirred for 2 hours prior to co-acrervation. BSA-dasatinib nanoshell was prepared over PLGA-mTHPC nanoparticles by ethanol co-acervation, in that 1:2 volume of absolute ethanol was added drop wise (l00ul/min) under stirring (800-1000 rpm) till the solution became turbid, to aid optimum albumin-dasatinib co-acervate coating over the PLGA-mTHPC NPs. The system is then added with 35ul of 4mg/ml l-Ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC) and stirred for 2 hours to link and harden the coacervates formed. The core-shell nanomedicine constructs were collected by lyophilizing the above solution.

Example 2: Polymer core /polymer shell photo-chemo-nanomedicine (PCL-mTHPC core/Chitosan-Sorafenib shell)

In this example preparation of a combinatorial polymer-polymer core-shell nanomedicine with photsensitizer mTHPC loaded Polycaprolactone (PCL) nanocore and dasatinib encapsulated chitosan shell is presented. ImM mTHPC containing polymer solution was prepared by dissolving the required 4mg/ml stock in 1.0 wt % PCL solution in chloroform and was stirred for 1 hr in dark prior to electrospray. This solution was then electrosptrayed at a rate of 1 ml/hr to a grounded glass beaker kept at a distance of 7cm that containing de-ionized water with 0.00 lv/v Tweeen 20® as surfactant and was maintained in constant stirring (600 rpm). The electrospray was carried out under ambient temperature, pressure and 60±5% humidity, by applying a potential of 1.3kV/cm, using a high voltage supply. The particles were acquired by lyophilizing the electrosprayed solution. The lyophilized particles are then mixed with 0.5% chitosan solution containing 5mM sorafenib. 0.25 wt% Tween 80 was added to his solution to prevent particle aggregation and the system was well mixed by stirring for 30 min. Chitosan-sorafenib nanoshell was prepared over PCL-mTHPC nanopaticles by ionic gelation process. Aqueous tripolyphosphate (TPP: 0.25% w/v) solution was added dropwise into the above solution and stirred under room temperature. The core shell nanoparticles were obtained by centrifuging the suspention at 12,000 rpm for 30 min

Claims:

1. We claim a core-shell photo-chemo nanomedicine system having a polymer core with average size of < 500nm and a shell made up of another polymer or protein with average size of < 500nm, and the core and the shell are loaded with a photosensitizer drug and chemo-drug, separately with distinct interface.

2. The composition of claim 1, wherein the polymer used is a single or blended cross linked polymer and the protein used is in its native or denatured and cross linked form.

3. The composition of claim 1, wherein the polymer core is loaded with at least one photosensitizer or chemo-drug

4. The composition of claim 1, wherein the polymer/protein shell is loaded with at least one chemo- or a photosensitizer-drug

5. The composition of claim 1, wherein the polymer used is preferably a natural or synthetic biodegradable polymer at least one from the group, but not limited to poly glycolic acid (PGA), poly(lactic-co-glycolic acid) (PLGA), glycolide/trimethylene carbonate copolymers (PGA/TMC); poly-lactides (PLA), poly-L-lactide (PLLA), Poly-DL-Iactide (PDLLA), L-lactide/DL-lactide copolymers; lactide/tetramethyl-glycolide copolymers, poly-caprolactone (PCL), poly-valerolacton(PVL), poly-hydroxy butyrate (PHB), poly vinyl alcohol (PVA) poly-hydroxy valerate(PHV), polyvinylpyrrolidone (PVP), Polyethyleneimine (PEI) and lactide/trimethylene carbonate copolymers, chitosan, carboxymethyl chitosan, chitin, pollulan, etc., or blends thereof.

6. The composition of claim 1, wherein the protein is chosen from, but not limited to the group of human serum albumin, or protamine, transferrin, lactoferrin, fibrinogen, gelatin, mucin, soy protein, apoferritin, ferritin, lectin, lactoferrin, gluten, whey protein, prolamines such as gliadin, hordein, secalin, zein, avenin, or combinations thereof.

7. The composition of claim 1, wherein the chemo drug loaded to the polymer core or polymer/protein shell is chosen from, but not limited to the group of anti-neoplastic agentssuch as cyclophosphamide, methotrexate, 5-fluorouracil, doxorubicin, cisplatin, hydroxyurea, leucovorin calcium, tamoxifen, flutamide, asparaginase, altretamine, mitotane, procarbazine hydrochloride, mechlorethamine, thioguanine, carmustine, lomustine, temozolomide, melphalan, chlorambucil, streptozocin, methotrexate, vincristine, bleomycin, vinblastine, vindesine, dactinomycin, 6-MP,daunorubicin, Lenalidomide, L-asparginase, doxorubicin, tamoxifen or anti-proliferative agents such as rapamycin, paclitaxel or anti-angiogenesis agents such as avastin, or inhibitors of tyrosine kinase including epidermal growth factor receptor (EGFR), vascular endothelial growth factor receptor (VEGFR), platelet derived growth factor receptor (PDGFR), fibroblast growth factor receptor (FGFR), Rous sarcoma oncogene/Breakpoint cluster region/Abl (Src-bcr-abl), Insulin-like growth factor 1 receptor (IGF-1R), FLT-3, HER-2, STAT5, c-Kit, c-Met, ALK, ETA receptor inhibitor, HIF inhibitor, Syk inhibitor, Tie2 kinase inhibitor and the like), Vascular disrupting agents (e.g. plinabulin), cell cycle/check point inhibitors like polo-like kinase (PLK) inhibitor (e.g. volasertib), cyclin dependent kinase (CDK) inhibitors (e.g. seliciclib, indirubin etc.,), topoisomerase inhibitors (e.g. adriamycin, camptothecin, etoposide, idarubicin, irinotecan, topotecan, mitoxantrone etc.,), microtubule inhibitors (e.g. docetaxel, paclitaxel, vincristine etc.,), antimetabolites (e.g. decitabine, gemcitabine, fludarabine etc.,), telomerase inhibitors, DNA & RNA replication inhibitors (e.g. clarithromycin, cytarabine, mitoxantrone HC1 etc.,)dihydrofolate reductase inhibitor, HDAC inhibitor, Bcl-2 and TNF-a inhibitors, PARP inhibitors, MAPK inhibitors, PI3K/Akt/mTOR inhibitors, integrase and protease inhibitors, Wnt/Hedgehog/Notch inhibitors, cAMP, lipide signaling inhibitors (e.g. PKC, PIM etc.,), TGF-P inhibitors, chemotherapeutic pro-drugs, antioxidant inhibitors like diethyl-dithiocarbamate, methoxyestradiol, 1-buthionine sulfoximine, 3-amino-l,2,4-triazole and the combinations thereof.

8. The composition of claim 1, wherein the photosensitizer loaded to the core or the shell is chosen from, but not limited to chlorine e6 (Ce6), meso-tetra(3-hydroxyphenyl)chlorin (m-THPC), methylene blue, benzoporphyrin derivative monoacid ring A (BPD or verteporfin), photofrin, Rose bengal, metal phthalocyanine, hypericin, toluidine blue O, pyropheophorbide-a hexyl ether (HPPH), Indium pyropheophorbide, padoporfin, padeliporfin, and combinations of thereof.

9. The composition of claim 1, wherein the chemo-drug is loaded into the core or the shell as monomers or aggregates or semi-aggregates or its combinations.

10. The composition of claim 1, wherein the photosensitizer is loaded into the core or shell as monomers or aggregates or semi-aggregates or its combinations.

11. The composition in claim 1, the photo-chemo nanomedicine will be able to deliver the drugs from the shell and the core sequentially or simultaneously

12. The composition of claim 1, the photo-chemo nanomedicine is specifically delivered to the disease like cancer either by passive or active targeting mechanisms

13. The composition in claim 12, where the active targeting is achieved by conjugating with ligands such as monoclonal antibody, (for example, CD20, CD33, CD34, CD38, CD44, CD47, CD52 CD90, CD123, CD133, EGFR, PDGFR, VEGF, HER2, Transferrin receptors and like) peptides, (for example RGD, CRGD, LyP-1 peptide, bombesin (BBN) peptide, FSH33, truncated human basic fibroblast growth factor peptide (tbFGF), octreotide etc.,), small molecules (folid acid, mannose, hyaluronic acid (HA)) , proteins (transferrin, somatoatatin) or aptamers and like.

14. W composition of claim 1, the photo-chemo nanomedicine is administered either intravenously, orally, parenterally, subcutaneously or by direct local delivery.

15. W claim a method of treatment using photo-chemo nanomedicine by applying chemotherapy followed by photodynamic therapy or photodynamic therapy followed by chemotherapy or simultaneous photodynamic- and chemo-therapy.

16. We claim a process for making core-shell photo-chemo nanomedicine loaded with a photosensitizer and chemo-drugs, either in the core or the shell

17. The process in claim 16, where the polymer core is first formed by a method of spontaneous emulsification or solvent diffusion or salting out or emulsification-diffusion or supercritical fluid technology or micro emulsion or double emulsion or electrospray or electro deposition or ultrasonication or spray drying or like.

18. The process in claim 16, wherein the protein shell is formed over the polymer core by simple desolvation or co-acervation, complex co-acervation, nano-precipitation, sol-gel, spray drying slating-out or cross linking

19. The process in claim 14, wherein the polymer shell over polymer core is formed by the process of nano-precipitation, microemulsion, spontaneous emulsification, double emulsion, emulsion-droplet coalescence, solvent evaporation emulsification, electrospray, solvent diffusion, salting out or emulsification-diffusion or supercritical fluid technology electro deposition or ultrasonication or spray drying or like.