WO2017115381A1

WO2017115381A1 - Degradable or transformable gold coated liposomal nano-construct and a process for its preparation

Info

Publication number: WO2017115381A1
Application number: PCT/IN2016/000296
Authority: WO
Inventors: Rohit Srivastava; Rinti Banerjee; Aravind Kumar RENGAN; Arpan PRADHAN; Abhijit De; Amirali Bakarali BUKHARI
Original assignee: Indian Institute Of Technology, Bombay; Tata Memorial Centre
Priority date: 2015-12-29
Filing date: 2016-12-27
Publication date: 2017-07-06

Abstract

The present invention provides a degradable or transformable gold coated liposomal nano- construct which is multifunctional. More particularly, the present invention provides a degradable nano-construct comprising gold coated lipid liposome, wherein said lipid liposome is made up of phosphatidylcholine and cholesterol and wherein said nano-construct is degraded either by enzyme, increase in temperature or photo-thermal. The present invention also provides a transformed nano-construct comprising gold coated lipid liposome, wherein said lipid liposome is made up of phosphatidylcholine and cholesterol and co-entraps at least one drug and wherein said nano-construct is in a dendrimer form, branched form or mesh form. Further, the present invention provides a process for preparing the afore- mentioned degradable and/or transformed nano-construct.

Description

DEGRADABLE OR TRANSFORMABLE GOLD COATED LIPOSOMAL NANO- CONSTRUCT AND A PROCESS FOR ITS PREPARATION The present application claims priority from Indian patent application no. 4910/MUM/2015, "DEGRADABLE OR TRANSFORMABLE GOLD COATED LIPOSOMAL NANO- CONSTRUCT AND A PROCESS FOR ITS PREPARATION", filed on 29 December 2015, the whole content of which is hereby incorporated for reference. FIELD OF THE INVENTION

The present invention relates to a degradable or transformable gold coated liposomal nano- construct which is multifunctional. More particularly, the present invention relates to a degradable nano-construct comprising gold coated lipid liposome, wherein said lipid liposome is made up of phosphatidylcholine and cholesterol and wherein said nano-construct is degraded either by enzyme, increase in temperature or photo-thermal. The present invention also relates to a transformed nano-construct comprising gold coated lipid liposome, wherein said lipid liposome is made up of phosphatidylcholine and cholesterol and co-entraps at least one drug and wherein said nano-construct is in a dendrimer form, branched form or mesh form. Moreover, the present invention relates to a process for preparing the afore- mentioned degradable and/or transformed nano-construct.

BACKGROUND OF THE INVENTION

Many organic and inorganic nanosystems are being actively researched for their efficiency in cancer diagnosis and treatment as well other clinical conditions. Among them, plasmonic nanostructures for photothermal therapy (PTT) has gained considerable importance owing to the advent of two ongoing PTT based clinical trials making use of gold nanoshells for the treatment of brain and metastatic lung tumors. These plasmonic nanostructures also serve as efficient candidates for imaging, drug delivery and thereby bringing out their multifunctional capabilities. According to the Food and Drug Administration (FDA) guidelines, any imaging agent (administered into the body) should be capable of getting cleared completely from the body within a reasonable period of time.

Gold-based materials deployed in PTT are generally larger than 20 nm in size. Accumulation of such metallic nanoparticles in the body could serve as a potential health risk. In 2013, Melnik et al. [Melnik, E. A.; Buzulukov, Y. P.; Demin, V. F.; Demin, V. A.; Gmoshinski, I. V.; Tyshko, N. V.; Tutelyan, V. A. Acta Nat. 2013, 5, 107-115] reported the transfer of silver nanoparticles via placenta to the rat fetuses, bringing out the gravity of risk involved in nanoparticle accumulation. Although many of such materials could serve as efficient imaging agents, their larger size and non-degradable nature prevents renal clearance, thus limiting their application. To achieve renal clearance the size of inorganic nanoparticles will have to be <5.5 nm. Inorganic, metal-containing nanoparticles less than 5.5 nm in size are capable of getting filtered through the glomerular basement membrane (GBM), thereby serving as ideal candidates for imaging (with renal route of clearance), but are unsuitable for PTT. Hence, a multifunctional nanosystem capable of achieving good body clearance through both hepato- biliary and renal route in addition to serving as effective agents for PTT is warranted.

Biodegradable plasmon resonant nanoparticles employing l,2-dipalmitoyl-j«-glycero-3- phosphocholine (DPPC) gold hybrid nanostructures were synthesized by Troutman et al. in 2008 [Troutman, T. S.; Barton, J. K.; Romanowski, M. Adv. Mater. 2008, 20, 2604-2608.]. The transition temperature of DPPC being 41 °C restricts its use only to drug delivery rather than hyperthermic killing of cancer cells (as biological cells begin to die of hyperthermia only when the temperature reaches >43 °C).

The present invention focuses on a degradable or transformable gold coated liposomal nano- construct having liposome-gold nanoparticle hybrid system that has such multifunctional capabilities. Because the core of this nano-construct is made up of biodegradable lipid, the gold coating on the surface is capable of splitting into smaller particles (<5-8 nm), and achieving both hepatobiliary and renal clearance. The surface gold could also get transformed into dendrimers when subjected to either enzymatic or mechanical degradation. Such nanoparticle systems also have an added advantage of getting accumulated specifically at the tumor site due to the leaky vasculature of developing blood vessels and poorly developed lymphatics (enhanced permeation and retention, EPR effect). In other words, they could be passively targeted to the tumor region. Alternatively, spatiotemporal control by external trigger is another form of achieving specificity, wherein drug delivery is controlled to a specific region by optical, magnetic, or ultrasound modalities. In contrast to the passive mode, active targeting involves antibody or affibody conjugation that binds with specific antigen at the tumor site. However, the added bulk of the antibody protein size and the cost of antibody limits its deployment on a larger scale. To overcome this limitation of increased cost factor, the spatiotemporal control of external trigger is an efficient and economical alternate to the antibody mediated targeting.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides a degradable nano-construct comprising gold coated lipid liposome, wherein said lipid liposome is made up of phosphatidylcholine and cholesterol and wherein said nano-construct is degraded either by enzyme, increase in temperature or photo-thermal. In another aspect, the present invention provides a transformed nano-construct comprising gold coated lipid liposome, wherein said lipid liposome is made up of phosphatidylcholine and cholesterol and co-entraps at least one drug and wherein said nano-construct is in a dendrimer form, branched form or mesh form. In yet another aspect, the present invention provides a process for preparing the aforementioned degradable and/or transformed nano-construct comprising gold coated lipid liposome. The process comprising the steps of:

a) Dissolving distearoylphosphatidylcholine (DSPC) and cholesterol in an organic solvent to form a solution;

b) Drying the solution in a rotary evaporator under vacuum to obtain a thin lipid film at 40°C;

c) Hydrating the lipid film with a buffer solution by rotating the round bottom flask at about 110 rpm to 150rpm at 60°C to form a homogenous suspension of multilamellar liposomes;

d) Sonicating the suspension of step (c) 5 minutes to 10 minutes to obtain small unilamellar liposomes; and

e) Adding chloroauric acid to the liposomes of step (d) followed by addition of ascorbic acid to form the gold coated lipid liposome nano-construct.

In a further aspect, the present invention provides a kit comprising either the afore-mentioned degradable nano-construct or transformed nano-construct.

Further scope and applicability of the present invention will become apparent from the detailed description given hereinafter. However, it should be understood that the detailed description and specific examples, while indicating embodiments of the present invention, are given by way of illustration only, because various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed.

ADVANTAGES OF THE PRESENT INVENTION

• The nano-construct of the present invention has multifunctional capabilities and can serve as a potential candidate for imaging, photothermal therapy, drug delivery as well as other therapeutic purpose.

• Both the gold and lipid liposome of the nano-construct are biocompatible and thus would not cause toxicity issues.

• The nano-construct of the present invention is in solution form and thus can be~ administered at the target site directly. This eliminates the need for surgical interventions.

• The aforementioned nano-construct can be prepared in a cost effective manner and can be easily scaled up.

• The aforementioned nano-construct releases the drug in a controlled and sustained manner.

• The aforementioned nano-construct may be used to treat superficial tumors/diseases (e.g. cervical intra epithelial neoplasia, cervical carcinoma, oral cancer, malignant melanoma, acne).

• The aforementioned nano-construct may also serve as intravenous mesh depot that can be used in venous grafts in bypass surgery thereby improving cardiac dynamics. It may also be used in treating Deep vein thrombosis DVT (that forms a major source for emboli formation), thereby preventing further complications like cardiovascular accident CVA and myocardial infarction MI.

• The aforementioned nano-construct may also be used in emergency medicine for localized adrenaline/urokinase delivery for better cardiac functioning.

BRIEF DESCRIPTION OF THE DRAWINGS The foregoing summary, as well as the following detailed description of the invention will be better understood when read in conjunction with the appended drawings. For the purpose of assisting in the explanation of the invention, there are shown in the drawings embodiments which are presently preferred and considered illustrative. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities shown therein.

Figure 1: Schematic diagram representing the principle of synthesis of degradable nano- construct (i.e. LiposAu NPs) and their mode of action to perform photothermal treatment causing DNA damage.

Figure 2: Characterization of degradable nano-construct (i. e. LiposAu NPs). (A) TEM image of LiposAu NPs. (Scale = 50 nm). (B) SEM image of LiposAu NPs. (Scale = 100 nm) (C) DLS size distribution of LiposAu NPs. (D) UV-vis absorbance spectra of lipase, liposome, and LiposAu NPs treated with lipase enzyme in comparison with LiposAu NPs (untreated). (E) HR-TEM analysis of LiposAu NPs indicating the presence of gold crystal lattice. (F) Temperature mediated LiposAu NPs degradation.

Figure 3: In vivo biodistribution and clearance of degradable nano-construct (LiposAu NPs). (A) TEM image of liver tissue showing (i) control hepatocytes and (ii), (iii) hepatocyte containing LiposAu NPs in aggregated state. (B) Mice plasma levels of ALT (U L) at the end of 24 h. (C) TEM images of kidney tissue showing (i) control tissue, (ii), (iii) kidney tissue containing LiposAu NP in its dissociated state with it less than 5 nm sized gold seeds. (D) Mice plasma values of creatinine (mg/dl) at the end of 24 h. (E) Graph represent tissue biodistribution of Au in vivo as determined by ICP-MS and ICPAES analysis at various end points. (F) ICP-MS based mice blood plasma levels of Au (ng/mL). (G) TEM image of blood plasma showing small gold particles of varying size range as represented in (i) and (ii). Significance designated as * indicates P < 0.05, ** indicates P < 0.005, and **** indicates P < 0.0001. Scale bar: A (i,ii) 2 μπι; A(iii) 1 μιη; C(i) 1 um; C(ii,iii) 20 nm; G(i) 5 nm; and G(ii) 2 nm. (H) ED AX analysis of liver tissue. (I) TEM and HR-TEM analysis of LiposAu NPs in liver and kidneys shows lattice arrangement. (J) In vivo tracking of LiposAu NPs labeled with Indocyanine Green (ICG) at varying time intervals. Free ICG was used as a vehicle control. Organs/tissue has been marked tentatively as indicated. T: Tumor; G: Gut; L: Liver; B: Bladder is marked in the figure by the green arrows. (K) ICP-MS analysis of the tumor tissue harvested 48 hours following NP injection indicates no specific uptake of gold. (L) Protein and blood analysis in mouse urine using dipsticks. (M) Standard curve of Bovine Serum protein and (N) Percent unbound protein after incubation with particles. (O) Graph showing percent hemolysis. (P) Morphology of RBC after incubation with particles for 2h. (Q) In vitro biocompatibility on NIT-3T3 cell line.

Figure 4: In vitro photothermal ablation of cancer cells by degradable nano-construct (LiposAu NPs). (A) Fluorescence micrograph images of photothermal therapy mediated cell death in MCF-7 -flue 2 -turboFP cancer cell line. Red color represents the fluorescence of the turboFP (635 nm emission) protein. (B) Quantitative analysis of bio luminescence based photothermal cell death in MCF-7 -fluc2-turboFP (P = 0.0034) and

(P =0.0024) cancer cells. Representative images for qualitative assessment are given below the graph representing the various groups. Pseudocolor bar indicates the photons captured by the CCD camera. (C) Representative images showing the formation of γΗ2Α.Χ foci after treatment in MCF-7 and HT1080 cancer cells. (D) Quantitative assessment of γΗ2Α.Χ foci in MCF-7 (P < 0.0001) and HT1080 (P = 0.0007) cancer cells. (E) Optimization of laser irradiation time on MCF-7~fluc2-turboFP and HTl080-fluc2-turboFP cancer cells. (F) (a) In vitro photothermal therapy on SiHa cells, (b-e) Qualitative microscopic images: (i) Bright field images of (b) control cells only (c) cells irradiated for 4min with 750nm laser (d) cells incubated with LiposAu (e) cells incubated with LiposAu and irradiated for 4 min with 750nm laser, (ii) corresponding dark field images (at 10X) of the same exhibiting PI stain (dead cells) in red.

Figure 5: In vivo temperature rise and particle concentration optimization by subcutaneous bleb formation.

Figure 6: In vivo photothermal ablation by LiposAu NPs in tumor xenograft. (A) Representative pre- and post-treatment in vivo bioluminescence images of mice bearing

tumor xenografts. (B) Quantitative assessment of bioluminescence to demonstrate the increase in tumor volume. The highlighted region indicates the treatment period (* indicates P < 0.05 and ** indicates P < 0.01). (C) Kaplan-Meier survival curve of the tumor bearing mice (P = 0.003). (D) Representative photographic and bioluminescence image of LiposAu NPs and laser treated mouse post 6 months of treatment reveals no signs of regression. (E) Bar diagram represents the fold change in bioluminescence between the laser, and LiposAu NPs + laser treated tumors. (F) Hematoxylin and Eosin (H&E) stained histological evaluation of tumor tissue after PTT.

Figure 7: Schematic of Lipos Au Mesh Depot preparation through mechanical degradation.

Figure 8: Transmission electron micrographs of A) Gold nano mesh depot (AuNMD), B) and C) Mechanically Degraded Au NMD through probe sonication for 10 min and 20 min respectively. Figure 9: Transmission electron micrographs of Lipos Au Mesh depot obtained by enzymatic (lipase) degradation.

Figure 10: Scanning electron micrographs of Lipos Au Mesh depot obtained through mechanical degradation.

Figure 11: A) HR-TEM showed lattice arrangement in Lipos Au mesh depot sample. B) ED AX analysis of Lipos Au mesh depot.

Figure 12: Model drug release profile of transformed nano-construct i.e. transformed Au NMD.

DESCRIPTION OF THE INVENTION

In describing the invention, the following terminology will be used in accordance with the definitions set forth below. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described herein. As used herein, each of the following terms has the meaning associated with it in this section. Specific and preferred values listed below for individual process parameters, substituents, and ranges are for illustration only; they do not exclude other defined values or other values falling within the preferred defined ranges. All publications mentioned herein are incorporated by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. As used herein, the singular forms "a," "an," and "the" include plural reference unless the context clearly dictates otherwise. The terms "preferred" and "preferably" refer to embodiments of the invention that may afford certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the invention.

When the term "about" is used in describing a value or an endpoint of a range, the disclosure should be understood to include both the specific value or end-point referred to.

As used herein, the terms "comprises", "comprising", "includes", "including", "containing", "characterized by", "having" or any other variation thereof, are intended to cover a nonexclusive inclusion.

As used herein, the term "nano-construct" refers to a construct whose size is in nanometer range. The size can vary from 5 nm - 400nm.

As used herein, the term "liposome" refers to a self-assembled spherical vesicle consisting of aqueous core surrounded by a lipid bilayer.

In one aspect, the present invention provides a degradable nano-construct comprising gold coated lipid liposome, wherein said lipid liposome is made up of phosphatidylcholine and cholesterol and wherein said nano-construct is degraded either by enzyme, increase in temperature or photo-thermal. Preferably the nano-construct of the present invention is degraded by lipase enzyme. In an embodiment of the present invention, the phosphatidylcholine is a phosphatidylcholine having a transition temperature (T_m) > 37°C. The exemplary phosphatidylcholine may be selected form the group consisting of distearoylphosphatidylcholine (DSPC), dipalmitoylphosphatidylcholine (DPPC) or mixtures thereof. Phosphatidylcholine and cholesterol are present in the weight ratio of 8:2 to form the lipid liposome. In an embodiment of the present invention, gold used for coating lipid liposome may be elemental gold or its oxides or salts thereof. The gold used in the present invention is chloroauric acid trihydrate, choloroauric acid tetrahydrate etc. Gold coated on the lipid liposome may be either partially coated or entirely coated.

In an embodiment of the present invention, the gold coated lipid liposome optionally comprises a drug or a dye. Suitable drug includes anti-cancer agent, antibacterial agent, antifungal agent, antiviral agent, anti-inflammatory agent, antibody, anti-microbial agent, non-steroidal anti-inflammatory agent or combinations thereof. Non-limiting examples of the drug may be doxorubicin, paclitaxel, vinblastine, colchicine, vincristine, vindesine, vinorelbine or combinations thereof.

Non-limiting examples of the dye includes ICG, IR780, IR820 or IR825.

The degradable nano-construct has a size range of 5 to 150 nm and the nano-construct of the present invention is nanoshells. The nano-construct of the present invention is thermo- responsive at above 42°C. The nano-construct of the present invention is also Near Infra-Red (NIR) responsive and is tuned at a wavelength of about 650 nm to about 915 nm.

In another aspect, the present invention provides a transformed nano-construct comprising gold coated lipid liposome, wherein said lipid liposome is made up of phosphatidylcholine and cholesterol and co-entraps at least one drug and wherein said nano-construct is in a dendrimer form, branched form or mesh form, preferably the nano-construct is in a dendrimer form. The nano-construct is transformed by enzymatic action or mechanical action, preferably by lipase enzyme, ultrasound, probe sonication or defibrillation.

In an embodiment of the present invention, the phosphatidylcholine is a phosphatidylcholine having a transition temperature (T_m) > 37°C. The exemplary phosphatidylcholine may be selected form the group consisting of distearoylphosphatidylcholine (DSPC), dipalmitoylphosphatidylcholine (DPPC) or mixtures thereof. Phosphatidylcholine and cholesterol are present in the weight ratio of 8:2 to form the lipid liposome. In an embodiment of the present invention, gold used for coating lipid liposome may be elemental gold or its oxides or salts thereof. The gold used in the present invention is chloroauric acid trihydrate, choloroauric acid tetrahydrate etc. Gold coated on the lipid liposome may be either partially coated or entirely coated.

Suitable drug includes anticoagulant, anti-cancer agent, antibacterial agent, anticholinergic, antifungal agent, antiviral agent, anti-inflammatory agent, antibody, hormone, enzyme, statin, anti-microbial agent, non-steroidal anti-inflammatory agent or combinations thereof. Non- limiting examples of the drug may be rifampicin, adrenaline, atropine, streptokinase, urokinase, heparin or combinations thereof.

In an embodiment of the present invention, the transformed nano-construct has increased drug release profile at 45 °C and releases the drug in a sustained manner. The transformed nano-construct has a size range of 5 to 150 run. In an embodiment of the present invention, the transformed nano-construct may be Near Infra-Red (NIR) responsive.

The afore-mentioned nano-constructs are biocompatible. The present invention also provides a kit comprising the degraded nano-construct and/or the transformed nano-construct as mentioned above.

In yet another aspect, the present invention also provides a process for preparing degradable and/or transformed nano-construct comprising gold coated lipid liposome, wherein said process comprising the steps of:

c) Hydrating the lipid film with a buffer solution by rotating the round bottom flask at about 110 rpm to 150 rpm at 60°C to form a homogenous suspension of multilamellar liposomes;

d) Sonicating the suspension of step (c) for 5 minutes to 10 minutes to obtain small unilamellar liposomes; and e) Adding chloroauric acid to the liposomes of step (d) followed by addition of ascorbic acid to form the gold coated lipid liposome nano-construct.

The organic solvent is selected from the group consisting of chloroform, methanol or mixtures thereof; preferably the organic solvent is a mixture of chloroform and methanol and is added in a ratio of 2: 1.

The buffer solution is selected from the group consisting of phosphate buffered saline (PBS), or simulated body fluid (SBF).

In an embodiment of the present invention, the liposome suspension of step (c) is sonicated using a probe sonicator with 40 - 50 % intensity. The chloroauric acid, liposome and ascorbic acid in step (e) are added in the ratio of 1 :2:4. In an embodiment of the present invention, said nano-construct of step (e) may be degraded either by enzyme, increase in temperature or photo-thermal or the nano-construct of step (e) may be transformed by lipase enzyme, ultrasound, probe sonication or defibrillation.

The afore-mentioned nano-construct (degradable and transformed) is stable and may be administered via intravenous, intratumoral, intramuscular, topical routes. It may be used for conditions such as but not limited to cancer therapy, topical skin diseases. It may be used as an adjuvant to drug eluting stent or as a cardiac conduction enhancer etc.

EXAMPLES

1. Materials and methods

Tetrachloroauric acid trihydrate (HAuCl₄.3H₂0) was purchased from Acros Organics (Thermo Fisher Scientific Inc., Belgium). Distearoyl phosphatidylcholine (DSPC) with >99% purity was obtained from Lipoid (Germany) and used without further purification. Cholesterol (CH) was purchased from Sigma Aldrich Company (St. Louis, USA). L- Ascorbic acid (AA) was purchased from SRL Pvt. Ltd, India. Dulbecco's Modified Eagle Medium (DMEM), RPMI 1640, Fetal Bovine Serum (FBS), antibiotic antimycotic solution, Phosphate Buffered Saline (PBS), and trypsin-EDTA solution were purchased from Life Technologies. All other reagents were purchased from Spectrochem India Pvt. Ltd. All chemicals were reagent grade and used as received. All glassware was cleaned with freshly prepared aquaregia and rinsed with water before use. A MilliQ water system (Millipore, Bedford, MA, USA), supplied with distilled water, provided high purity water for these experiments. 2. Preparation of Thermo-sensitive Liposomes

The liposomes were prepared by the thin film hydration method. Briefly, mixture of lipids DSPC and cholesterol with different molar ratio was dissolved in 2:1 chloroform: methanol mixture. The solution was then dried in a rotary evaporator under vacuum to obtain a thin lipid film at 40°C. The lipid film was hydrated with a phosphate buffer saline solution by rotating the round bottom flask at about 180 rpm at 60°C until the lipid film was completely hydrated and a homogeneous dispersion was formed. The liposome suspension was then sonicated for 3 cycles with 40% to 50% intensity using probe sonicator (each sonication cycle was performed for 2 minutes with 2 second on/off pulse) to obtain small unilamellar liposomes.

3. Preparation of Gold Coated Liposomes

Liposomes made up of DSPC: CHOL with molar ratio 8:2 with concentration 2mg/ml were prepared with the thin film hydration method. After preparing multi lamellar vesicles (MLVs), they were sonicated to obtain unilamellar liposomes. ΙΟΟμΙ of 2.5mM HAuCl₄.3H₂0 was added to 200μ1 of liposome solution (2mg/ml lipid concentration), followed by 400μ1 of ascorbic acid AA (5mM), which produced an abrupt color change from the characteristic translucent white of liposomes to a greenish blue color solution. They were further characterized by Dynamic Light Scattering (DLS) - (BI 200SM, Brookhaven Instruments Corporation, USA), Transmission Electron Microscopy TEM (HR-TEM - JEM 2100, JEOL 2100F - 200 kV, FEI Technai 12 BioTwin-120 kV), and Scanning Electron Microscopy, SEM (JSM-7600F).

4. Serum protein adsorption study

For any formulation delivering intravenous, serum protein interaction is to be investigated. For this study Fetal Bovine Serum (FBS) was used after diluting it 10 times and incubated it with the Lipos Au NPs (4mg/ml) for different time span (1, 2, 3.5 and 5 h). After incubation particles were recovered by centrifugation and supernatant was taken for estimation of remaining protein. 5. RBC Stability

Blood sample to study the stability of RBC was diluted 50 times with 0.9% saline (pH=7.4) so as to obtain the significant difference and prevent any noise in reading the data which usually occurs at high concentration. To perform the RBC stability assay 190μ1 of blood sample was taken in 96 wells plate to this ΙΟμΙ of LiposAu NPs was added in different dilutions (1, 20, 100, 800, 2000 μg/ml) and each dilution was triplicated. Negative control (NC) contain ΙΟμΙ of PBS (pH=7.4) instead of testing sample so theoretically there should be no lysis of RBC in negative control. In positive control (PC) ΙΟμΙ of 20% triton-X was taken in place of testing sample, this leads to complete lysis of RBC. Once all the samples were added plate was incubated at 37°C for 2h and reading was taken after incubation. Percent hemolysis was calculated using follo

Reading was taken using filters at 405nm, 450nm, and 560nm because hemoglobin gives absorption at these wavelengths; all these three wavelengths have their own significance 405nm is preferred when lysis of cells is more, 450nm is used for general purpose, and 560nm is used when lysis of cells is less. So here I have preferred optical density at 560nm because lysis of cells is comparatively less. To see if there are any morphological changes taken place; After incubation with particles microscopic images of RBC were taken which were collected by centrifugation, by staining with eosin dye.

6. Cell Viability Assay

In vitro biocompatibility study was performed using the Alamar Blue assay on NIH 3T3 cell line. Exponentially growing cells were dispensed into a 96 well plate at a concentration of 1 x 10⁴ cells per well. After allowing 24 hours for cell attachment, LiposAu NP solutions were diluted appropriately in fresh media and added in the lipid concentration ranging from 125μ¾^/ηι1 to lmg/ml in triplicates. The media was not changed during the incubation period of 48 hours. Following incubation, cell viability was determined by the addition of Alamar Blue (20 ml, 1 mg/ml dye in sterile PBS). The plate was incubated for an additional 4 hours at 37°C and 5% C0₂, allowing viable cells to convert the blue solution into pink dye. Absorbance values at 560 nm and 620 nm were collected and cell viability was calculated as a percentage compared to untreated control cells. For bioluminescence based cell viability monitoring, 1 x 10⁵ cells were seeded per well in a black 96 well plate (Corning, USA) in triplicates in four groups (control, LiposAu NPs, laser only, and LiposAu NPs + laser). Next day, the LiposAu NPs group and LiposAu NPs + laser group were incubated with LiposAu NPs for 6 hours. The laser group and the LiposAu NPs + laser group were irradiated with NIR laser for a period of 4 minute per well and the readings were measured after 24 hours. 50μ1 of D-luciferin substrate (Biosynth, Switzerland) was added prior to measurement. The bioluminescence light output was captured using the IVIS Lumina II (Caliper Life Sciences, USA) imaging system. The exposure time was set to 5 seconds and readings were quantified using the Living Image v4.4 software. The light output was represented in terms of average radiance (photons/seconds/cm /steradian).

In vitro photothermal therapy was also performed on the SiHa cervical cancer cell line. SiHa cells were cultured in DMEM media. Such exponentially growing cells were seeded into a 96-well plate with a density of l x lO⁴ cells per well one day before the laser irradiation experiment. After 24 hours, cells were properly washed with PBS. Four sets of reactions were arranged - three controls namely control cells only, cells incubated with LiposAu NPs, cells receiving laser treatment (5 mins) and the last group comprised of cells receiving a combination treatment of LiposAu NPs and laser. The experiment was carried out in triplicates. After adding ΙΟΟμΙ of gold coated liposomes (4mg/ml lipid concentration) per well, cells were incubated at 37°C, 5% C0₂ for 5 hours. After 5 hours, the respective wells are irradiated with a 750nm laser (650mW-PMC, India). All the controls were irradiated for 5 min (max. irradiation time). The liposAu + laser groups were irradiated for 2 mins, and 5 mins. The plates were again incubated for another 12 hours at 37°C. Next day, cells were washed with PBS to remove unbound particles and subjected to MTT assay. For qualitative analysis, another set of 96 well plates were seeded with a density of 1 ^χ 10⁴ cells per well. The protocol followed for qualitative study remains the same as the one mentioned above but with a slight variation. At the end of 5 hours, cells were washed with PBS and PI dye was added (ΙΟΟμΙ per well). The cells were visualized with a fluorescence microscope equipped with filters set for excitation/emission wavelengths at 488/617nm for PI dye (red color indicating dead cells) at 10X magnification.

7. Immunofluorescence 5x10⁵ cells were plated on nitric acid pre-treated coverslips 48 hours prior to immunofluorescence. Next day, the cells were washed 3 times with PBS and incubated with LiposAu NPs for 6 hours. Prior to incubation, the respective groups were irradiated with NIR laser (650m W, PMC, India) for 4 minutes. After 1 hour of treatment, cells were then fixed with 4% paraformaldehyde (Sigma Aldrich, USA) for 10 minutes at 37°C. The cells were then permeabilized in 4% paraformaldehyde with 0.2% Triton X-100 (Sigma Aldrich, USA) for 10 minutes at room temperature. Cells were again washed 3 times with PBS and blocked with 2% BSA in PBS for 30 minutes at room temperature. Cells were incubated with the a- phospho-H2A.X primary antibody in a humidified chamber overnight at 4°C. Cells were washed 3 times with PBS on the next day and incubated with the respective dye labelled Daylight 633 secondary antibody. Nuclei were counterstained with DAPI ^g/ml). Coverslips were mounted using the VECTASHIELD mounting medium for fluorescence (Vector Laboratories Inc., CA). Images were captured using the Zeiss LSM 510 Meta Confocal Microscope (Carl Zeiss, Germany). Mean fluorescence quantitation was performed using the LSM Image Browser.

8. In vivo Biodistribution

The in vivo biodistribution was carried out as per the protocol approved by the Institutional Animal Ethics Committee (IAEC) of ACTREC and Vimta vide protocol (Protocol No. PCD/TDS/RAT/01). All ethical practice laid down in the Committee for the Purpose of Control and Supervision on Experiments on Animals (CPCSEA) guidelines for animal care were followed during the study. Test items were administered intravenously at a concentration of ~110μg/400μl to male mice that were divided as follows Gl - Normal saline (n=3), G2 - Liposome without Au (n=6), and G3 - LiposAu NPs (n=18; 6 per time interval). Blood was collected by retro-orbital plexus puncture in K2-EDTA coated tubes, urine was collected by direct .bladder puncture prior euthanasia, and major organ tissue samples were also collected. All collections took place at time intervals of day 1, 7 and 14 post-dosage for G3 group and at day 1 for Gl and G2 group. Plasma was separated by centrifugation and all the samples (including tissues, feces and urine sample) were utilized for further analysis.

9. In vivo Bioluminescence Imaging

The in vivo bioluminescence imaging was performed after obtaining clearance from the Tata Memorial Centre- ACTREC s Institutional Animal Ethics Committee (IAEC). 1 x 10⁶

cells were injected subcutaneously on the right flank of the BALB/c NUDE mice. Assessment of tumor implantation was carried out by imaging mice for 30 seconds using IVIS Lumina II and IVIS Spectrum imaging systems (Caliper Life Sciences, USA) after intra-peritoneal administration of D-luciferin (3mg/mouse) and then randomly segregated into control (n=5), laser control (n=5), and PTT treatment group (LiposAu NPs and laser) (n=5). Imaging was carried out at regular interval of 10 days to monitor the tumor growth. Intratumoral injections of 30μ1 normal saline and 30μ1 of (0.5μ§ μ1) LiposAu NPs were administered to the control and PTT treatment group respectively. 750 nm fixed wavelength laser irradiation was performed for 4 minutes on the tumors of the laser group and on the site of injection for the PTT treatment group. Pre and post treatment bioluminescence imaging was also performed to understand the effect of the treatment with respect to the ablation of tumor in vivo. The light output was quantified using the Living Image v4.4 software in terms of average radiance (p/sec/cm /sr). Pseudocolor bar represents the photons captured by the CCD camera. 10. Statistical Analysis

All statistical analysis was performed using GraphPad Prism 6 software (GraphPad Software, La Jolla California, USA). Error bars represent SEM. -values were calculated using student's t test and Log-rank (Mantel-Cox) test. P-values of < 0.05 were considered significant, and P- values of < 0.001 were considered highly significant.

11. Results for degradable nano-construct (i.e. LiposAu Nanoparticles)

LiposAu NPs were synthesized as per established protocol with slight modification to achieve a size range of 100-120nm. A schematic representing the synthesis and photothermal effect on LiposAu NPs including their ability to cause DNA damage and self-destruction achieving size reduction is shown in Figure 1. Representative transmission electron microscope (TEM) and scanning electron microscope (SEM) images of these LiposAu NPs have been shown in Figure 2A and 2B. Dynamic Light Scattering (DLS) measurement indicates a size range of about lOOnm (Figure 2C) and the polydispersity index as 0.18. The lattice arrangement of Au is clearly visible in High Resolution-TEM (HR-TEM) images of the surface region of these LiposAu NPs (Figure 2E). Such type of plasmon resonant nanoparticles has shown to be responsive for specific wavelength of laser light mediated excitation. Hence, the LiposAu NPs were tuned to an absorbance range of 750nm to achieve photothermal effect when subjected to a beam of 750nm laser light with 650mW power (Figure 2D). These LiposAu NPs when treated with lipase enzyme lost their NIR absorbance peak, confirming their degradable nature (Figure 2D). Also, when treated with specific temperature increments (water bath mediated), these LiposAu NPs showed corresponding reduction in NIR absorbance denoting their thermo-sensitivity (Figure 2F). The in vivo biodistribution and pharmacokinetic study was performed using Swiss albino mice. The analysis of various tissues, plasma, and urine was performed at varying time periods (Day 1, 7, and 14) after intravenous injection of LiposAu NPs (~ 110 μ g/400 μ L) through the tail vein. It was found that the majority of the injected particles were accumulated in the liver, followed by the spleen and kidney of the mouse. As these NPs were not targeted, they were directly taken up by the reticulo-endothelial system (i.e., liver and spleen) of the mouse. As the liver is the major metabolizing organ of the body playing an important role in lipid metabolism, the probability of LiposAu NPs to undergo enzymatic degradation gets maximized owing to their greater accumulation in the liver region. The accumulation and metabolic degradation of these NPs in the liver was qualitatively confirmed by TEM analysis of the liver tissue. As revealed from Figure 3A, 1 day after intravenous delivery, these NPs were found to be in an aggregated state, but their original spherical morphology was completely lost.

This indicates that under in vivo condition the LiposAu NPs present in systemic circulation would undergo metabolic degradation due to enzymatic activity in hepatocytes. Further, to confirm the aggregation observed in TEM, energy dispersive X-ray spectroscopy (EDAX) analysis was performed (Figure 3H). Inductively coupled plasma -atomic emission spectroscopy (ICP- AES) analysis of mouse liver revealed considerable accumulation of gold, right from day 1 end point analysis. But there was a considerable decrease in the %ID/g on subsequent long-term study. The observed reduction in the % ID from about 52% (day 1) to 9.8% (day 7) and further declined to about 3% (day 14) (P = 0.0037) (Figure 3E). The percentage reduction of Au in the liver at 14 days was further confirmed by TEM analysis (Figure 31). Also, HR-TEM images of liver and kidney samples confirm the presence of degraded Au NPs showing lattice arrangement that further indicates their metallic nature (Figure 31). The negligible Au values detected in the liver of the normal saline-treated controls were subtracted from those of the treated samples as background corrections. Similar observations were noted for the spleen, kidneys, and intestine. We also performed TEM analysis of blood plasma at 2 h end point that showed Au NPs of size 2 -8nm [Figure 3G(i),(ii)]. Presence of such small Au NPs in blood suggests that the disintegrated NPs (from the larger LiposAu NPs) reach the circulation after their enzymatic degradation in liver.

Table 1: ICP-MS analysis of Au in mice feces samples.

Table indicates the ICP-MS analysis ofAu in mice feces samples collected at 1, 7 and 14 day time intervals. The values obtained from the test animals were normalized with the average value of the control animals (0.000552±0.00011) to apply a correction factor.

Table 2: ICP-MS analysis of Au in mice urine samples.

Table indicates the ICP-MS analysis of Au in mice urine samples collected at 1, 7 and 14 day time intervals. The values obtained from the test animals were normalized with the average value of the control animals (8.37±0.71) to apply a correction factor.

It was observed that the majority of the injected particles are accumulated in the liver and spleen and the smaller percentage of 2 -8 nm particles circulating in blood could have resulted in accumulation in the kidney and further clearance through urine. TEM analysis of the kidney identified the presence of such smaller particles (Figure 3C). The inductively coupled plasma-mass spectroscopy (ICP-MS) analysis of kidneys indicated an accumulation of about 2.7% at day 1 that reduced to an approximate 0.25% at day 7 and further to about 0.22% on day 14 (Figure 3E). Though the current percentage of accumulation in the kidney is small in comparison to the liver we expect an improvement in renal clearance when the LiposAu NPs are subjected to both photothermal and enzymatic degradation. The current study has limited scope of understanding the real-time in vivo biodistribution and enzymatic degradation of LiposAu NPs. Though some amount of particles is getting cleared through the hepato-biliary route (Table 1), we find a small amount of Au in urine even on day 7 and 14 indicating the possibility of renal excretion (Table 2). We however speculate that the excretion of Au through the renal route is a constant process overtime and determination of this complete excretion in real time remains a limitation. Generally, individual small molecules like albumin get repelled by the negatively charged glomerular basement membrane (GBM) of the nephrons. This charge-based repulsion prevents any accumulation of negatively charged particle/molecule in the kidney that in turn restricts their excretion through urine. The higher accumulation of gold in the kidney at day 1 time period indicates that gold NPs owing to their positively charged surface were able to overcome the charge- based repulsion in the GBM. The ICP-MS analysis of mice plasma showed significant reduction of gold between day 1 and 14 end point analysis (P < 0.0001) (Figure 3F). The value of gold was reduced from 390± 13.7 ng/mL to 105.4 ± 5.1 1 ng/mL. As already pointed out, the size range of the gold NPs observed in mice blood plasma was also falling under the range of renal excretion. Also, the ICP-MS analysis of urine samples (collected at day 1, 7, and 14) revealed the presence of gold in varying concentrations confirming its excretion through the renal route (Table 2). Furthermore, we studied biodistribution in tumor-bearing mice using indocyanine green (ICG; NIR dye) coated LiposAu NPs to facilitate their short- term tracking in vivo. In order to understand if they possess any tumor homing properties after intravenous injection, we made use of the HT1080 xenograft model. Data suggests that these particles are not capable of any specific homing at the tumor site owing to their non- targeted nature. Also, in order to achieve successful homing several parameters play a key role, one such being the leaky vasculature of the tumor bed. However, unlike only ICG injected control mice, we were able to obtain considerable signal from the bladder region for up to a period of 24 h (Figure 3J) suggesting possible renal clearance. ICP-MS validations also confirm no LiposAu NPs uptake in the tumor (Figure 3K).

To understand the toxic effect of these gold NPs accumulation in liver and kidney, the blood plasma serum glutamic pyruvic transaminase (SGPT or ALT) and creatinine levels were also analyzed. No significant difference was observed between the control and the LiposAu NPs treated groups (Day 1) (Figure 3B, D) confirming that no specific acute toxicity to the liver or kidney was exhibited. Qualitative urine dipstick analysis was also performed in mice urine (LiposAu NPs treated and controls). There was no detectable blood or protein observed in the urine samples (Figure 3L). In vitro biocompatibility studies including serum protein adsorption demonstrated significant results (Figure 3N). On monitoring the protein adsorption, no significant adsorption of proteins on the particles was seen during the incubation of LiposAu NPs at 1, 2, 3.5, 5 hours with serum protein. Similarly RBC stability studies demonstrated positive findings (Figure 30, P). Biochemical analysis of hemolysis was found to be in correlation with that of morphological findings and either showed very little or negligible hemolysis (<0.5% at 80(^g/ml and 2.5% at concentrations as high as 2mg/ml).

In the absence of human data availability, such biodistribution studies involving experimental animals stands as the most reliable approach to determine the toxicity properties of chemically synthesized NPs. The close resemblance of anatomy and physiology of mice to that of humans enables us to predict the likely pharmacokinetics of these NPs in future human clinical transition. Additionally, in vitro biocompatibility on NIH-3T3 cells revealed no indications of toxicity associated with LiposAu NPs (Figure 3Q).

In vitro photothermal efficacy studies were performed using MCF-7 (breast) and HT1080 (fibrosarcoma) cancer cell lines. Both cell types were engineered for overexpressing a fusion reporter, that is, firefly luciferase 2 (fluc2) and turboFP fluorescent protein. Optimization of the laser irradiation time suggests a lethal effect on the breast cancer cells beyond 4 min of continuous exposure (Figure 4E). Hence, 4 min was chosen as the ideal irradiation time for a concentration of 15 μg/mL LiposAu NPs photothermal treatment. Qualitative assessment of the photothermal efficacy was studied in vitro using MCF-7 -flue 2 -turboFP cells by fluorescence microscopy. Combination treatment of LiposAu NPs and laser showed loss of fluorescence (suggesting cell death) at the area of contact as indicated by the arrow in Figure 4A. Such ablation of cancer cells is due to heat generation in the region of laser contact where LiposAu NPs are also present. Additionally, no change was observed in the fluorescence signal of either the untreated control or the internal control groups (LiposAu NPs only and laser only). This result was further supported by the significant reduction of fluc2 luminescence in MCY -l-fluc2-turboFP (P

0.0024) cells when compared to laser-treated control (Figure 4B). Herein, the luciferase light output is a direct measure of cell viability as the fluc2 enzyme catalyzes its substrate D- luciferin only in the presence of cellular ATP. As PTT is known to cause DNA damage mediated cell death. We also monitored the formation of γΗ2Α.Χ foci, a marker for DNA double strand breaks. Minimal or no γΗ2Α.Χ foci were observed in the untreated control and only LiposAu treated cells. The only laser- treated control cells revealed a slightly higher number of the γΗ2Α.Χ foci in comparison. However, it was seen that the cells that received the combination treatment with both LiposAu and laser, demonstrated the presence of highly significant number of γΗ2 A.X foci formations in both the MCF-7 (P < 0.0001) and HT1080 (P = 0.0007) cells (Figure 4C, D). This validates that the photothermal therapy mediated DNA double strand break was responsible for cancer cell ablation. Also, combination treatment of LiposAu NPs and laser in the SiHa cervical cancer cell line suggests a significant cell death at the 5 mins time period [Figure 4F (a)]. The significant increase in cell death for group that received the combination treatment of LiposAu NPs and laser was also confirmed by staining the cells with propidium iodide (indicator of dead cells) and qualitative images were captured using a fluorescence microscope [Figure 4F (b-e)].

In vivo temperature increment was critically determined by creating subcutaneous blebs of normal saline or LiposAu NPs in varying volumes (25, 50, and 100 μΐ) in hairless (BALB/c Nude) mice. Each of the blebs was treated with the NIR laser for 4 min. The temperature of the blebs was continuously monitored by an IR thermometer pre- and post-treatment. The blebs injected with normal saline did not show any specific temperature increment after 4min of continuous laser irradiation. However, the blebs injected with LiposAu NPs showed a temperature increment up to 7°C with 4 min of laser irradiation. There was also eschar formation on the treated area (noticed after 24 h of treatment), indirectly indicating temperature increment (Figure 5). Such eschar formation has been previously reported for gold nanoshells based PTT as well.

Further, in vivo photothermal efficacy was determined using Tl080-fluc2-turboFP tumor xenograft model in BALB/c NUDE mice. On the 20th day with growing tumors (average size of about 70 mm³), mice were randomly segregated into 3 groups (n = 5 per group). Group I received normal saline (30μL) as vehicle control; group II animals were treated with laser only while group III animals were given the combination treatment of LiposAu NPs (0.5 μg/μL in 30 μί) and laser. Group II and III animals were subjected to laser irradiation for a period of 4 min. The treatment cycle was divided into two rounds between day 20 and 30. Two days interval was kept between the treatment cycles to avoid any therapy burden on the animals. All animals were imaged by injecting D-luciferin substrate every 10th day starting from day 0 until the end of the experiment. It reveals a significant reduction in the bioluminescence signal (3.36 ^χ 10⁶ ± 1.52 ^χ 10⁶ p/sec/cm²/sr) on day 30 of the group III animals when compared to group I (5.47 ^χ 10¹⁰ ± 2.08 x 10¹⁰ p/sec/cm²/sr) (P = 0.0302) or the group II (4.95 ^χ 10¹⁰ ± 1.75 x 10¹⁰ p/sec/cm²/sr) (P =0.0068) animals (Figure 6Α,Β)· Additionally, the group III animals demonstrated complete regression of tumor and a 4 out of 5 animal survived until 6 months (P = 0.003) in contrast to the group I and group II animals, which all died within 35-40 days due to tumor burden (Figure 6C). At the end point of monitoring, noninvasive in vivo bioluminescence imaging of group III showed no signal output at the therapy site (Figure 6D). It was noted that the combination treatment of LiposAu NPs with laser results in a 4.63 fold reduction of the bioluminescence signal with respect to the respective controls (Figure 6E). Histopathological analysis revealed that the combination of LiposAu NPs and laser resulted in the most extensive necrotic response in that region. Because of this combination treatment, the tumor cells in the underlying region were completely ablated, whereas about 95% of the tumor mass remained in laser treated animals (Figure 6F).

12. Synthesis of Lipos Au mesh depot (transformed nano-construct)

Lipos Au mesh depot was prepared through either mechanical degradation or enzymatic degradation of lipos Au. In mechanical degradation process the lipos Au was sonicated with 40% amplitude for different time intervals (10 min, 15 min and 20 min) to get various branched structure of lipos Au mesh depot. On the other hand enzymatic degradation of lipos Au was performed using lipase enzyme. Lipase (1 mg/ml) was added to lipos Au solution and incubated at 37 °C for 24 h to get lipos Au mesh depot.

13. Characterization of Lipos Au mesh depot (transformed nano-construct)

13(a) Transmission electron microscopy (TEM)

TEM images of Lipos Au mesh depot were obtained using JEOL 21 OOF transmission electron microscope operating at 200 kV. Samples were deposited onto carbon coated copper grids, dried at room temperature.

13(b) Scanning electron microscopy

Lipos Au mesh depot was analyzed by using Scanning electron microscopy (SEM). Samples were then air-dried and observed using JSM-7600F Scanning Electron Microscopy. 13(c) In vitro release study

Liposomes loaded with model drug were prepared by previously reported methods with slight modification. 18 First the drug, which was not encapsulated, was removed from the liposome suspension by centrifugation at 20000g, 4°C for 20 min and then resuspended in PBS to make the total lipid concentration 2 mg/ml. This formulation of liposomes containing drug was entrapped in dialysis membrane and submerged in reservoir in which release was carried out at different temperature (32°C, 37°C and 45°C). For 100% release, liposomes were treated with 1% Triton X. Percentage release of drug was calculated by the following formula,

% Release= [A_t/ A_f] x 100

Where, A_t = Absorbance at time t,

A_f = Absorbance with 100% release, i.e. Triton-X treatment.

14. Results for transformed nano-construct

Liposome prepared by thin film hydration method. These were further coated with gold with aid of HAuCl₄ and ascorbic acid. This resulted in formation of Au nano mesh depot (Au NMD) as shown in Figure 8 A. This Au NMD was subjected to mechanical degradation using probe sonicator (30% amplitude, 2 sec. on/off pulse for varying time periods). This mechanical stress was expected to simulate the constant contraction and relaxation of heart/calf muscles that would aid in biotransformation of Au NMD. As shown in Figure 8B and C, the formation of dendrimer like stents (from Au NMD) was evident. Similar kind of dendrimer like stent structures were obtained upon degradation of Au NMD in the presence of lipase enzyme shown in Figures 9A, B and C. This indicates the possibility of biotransformation that could happen in situ under in vivo condition.

The scanning electron micrography (SEM) revealed the opening up of Au NMD to form dendrimer like stent structure as shown in (Figure lOA-lOC). HR TEM analysis revealed the lattice arrangement of Au (Figure 11 A) and ED AX analysis confirmed the presence of Au (Figure 11B).

15. In vitro release study

Model drug release experiment was performed to understand the drug release profile of the transformed nano-construct using rifampicin as a model drug. The drug release profile showed initial burst release for first 4 h followed by continuous sustained release for 30 h. The transformed nano-construct owing to its thermo responsive nature showed increase drug release profile at higher temperature (45°C). This could be used for photothermal mediated drug release at specific site using NIR laser (Figure 12).

Claims

CLAIMS:

1. A degradable nano-construct comprising gold coated lipid liposome, wherein said lipid liposome is made up of phosphatidylcholine and cholesterol and wherein said nano-construct is degraded either by enzyme, increase in temperature or photo- thermal.

2. The degradable nano-construct as claimed in claim 1, wherein said phosphatidylcholine is a phosphatidylcholine having a transition temperature (T_m) > 37°C.

3. The degradable nano-construct as claimed in claims 1 and 2, wherein said phosphatidylcholine is selected form the group consisting of distearoylphosphatidylcholine (DSPC), dipalmitoylphosphatidylcholine (DPPC) or mixtures thereof.

4. The degradable nano-construct as claimed in claim 1, wherein said phosphatidylcholine and cholesterol is present in the weight ratio of 8:2 to form the lipid liposome.

5. The degradable nano-construct as claimed in claim 1, wherein said gold is elemental gold or its oxides or salts thereof.

6. The degradable nano-construct as claimed in claim 1, wherein said gold is either partially or entirely coated on the lipid liposome.

7. The degradable nano-construct as claimed in claim 1, wherein said gold coated lipid liposome optionally comprises a drug or a dye.

8. The degradable nano-construct as claimed in claim 7, wherein the drug is selected from the group consisting of anti-cancer agent, antibacterial agent, antifungal agent, antiviral agent, anti-inflammatory agent, antibody, anti-microbial agent, non-steroidal anti-inflammatory agent or combinations thereof.

9. The degradable nano-construct as claimed in claim 8, wherein the drug is selected from the group consisting of doxorubicin, paclitaxel, vinblastine, colchicine, vincristine, vindesine, vinorelbine or combinations thereof.

10. The degradable nano-construct as claimed in claim 7, wherein the dye is selected from the group consisting of ICG, IR780, IR820 or IR825.

11. The degradable nano-construct as claimed in any of the preceding claims, wherein said nano-construct has a size range of 5 to 150 nm.

12. The degradable nano-construct as claimed in any of the preceding claims, wherein said nano-construct is a nano-shell.

13. The degradable nano-construct as claimed in any of the preceding claims, wherein said nano-construct is thermo-responsive at above 42°C.

14. The degradable nano-construct as claimed in any of the preceding claims, wherein said nano-construct is Near Infra-Red (NIR) responsive and is tuned at a wavelength of about 650 nm to about 915 nm.

15. The degradable nano-construct as claimed in any of the preceding claims, wherein said nano-construct is degraded by lipase enzyme.

16. The degradable nano-construct as claimed in any of the preceding claims, wherein said nano-construct is biocompatible.

17. A transformed nano-construct comprising gold coated lipid liposome, wherein said lipid liposome is made up of phosphatidylcholine and cholesterol and co-entraps at least one drug and wherein said nano-construct is in a dendrimer form, branched form or mesh form.

18. The transformed nano-construct as claimed in claim 17, wherein said phosphatidylcholine is a phosphatidylcholine having a transition temperature (T_m) > 37°C.

19. The transformed nano-construct as claimed in claims 17 and 18, wherein said phosphatidylcholine is selected form the group consisting of distearoylphosphatidylcholine (DSPC), dipalmitoylphosphatidylcholine (DPPC) or mixtures thereof.

20. The transformed nano-construct as claimed in claim 17, wherein said phosphatidylcholine and cholesterol is present in the weight ratio of 8:2 to form the lipid liposome.

21. The transformed nano-construct as claimed in claim 17, wherein said gold is elemental gold or its oxides or salts thereof.

22. The transformed nano-construct as claimed in claim 17, wherein said gold is either partially or entirely coated on the lipid liposome.

23. The transformed nano-construct as claimed in claim 17, wherein the drug is selected from the group consisting of anticoagulant, anti-cancer agent, antibacterial agent, anticholinergic, antifungal agent, antiviral agent, anti-inflammatory agent, antibody, hormone, enzyme, statin, anti-microbial agent, non-steroidal anti-inflammatory agent or combinations thereof.

24. The transformed nano-construct as claimed in claim 17, wherein the drug is selected from the group consisting of rifampicin, adrenaline, atropine, streptokinase, urokinase, heparin or combinations thereof.

25. The transformed nano-construct as claimed in any of the preceding claims, wherein said transformed nano-construct has increased drug release profile at 45 °C.

26. The transformed nano-construct as claimed in any of the preceding claims, wherein said transformed nano-construct releases the drug in a sustained manner.

27. The transformed nano-construct as claimed in any of the preceding claims, wherein said nano-construct is transformed by enzymatic action or mechanical action.

28. The transformed nano-construct as claimed in any of the preceding claims, wherein said nano-construct is transformed by lipase enzyme, ultrasound, probe sonication or defibrillation.

29. The transformed nano-construct as claimed in any of the preceding claims, wherein said nano-construct is biocompatible.

30. The transformed nano-construct as claimed in any of the preceding claims, wherein said nano-construct has a size range of 5 to 150 nm.

31. A kit comprising the degraded nano-construct as claimed in claims 1-16.

32. A kit comprising the transformed nano-construct as claimed in claims 17-30.

33. A process for preparing degradable and/or transformed nano-construct comprising gold coated lipid liposome, wherein said process comprising the steps of:

d) Sonicating the suspension of step (c) for 5 minutes to 10 minutes to obtain small unilamellar liposomes; and

34. The process as claimed in claim 33, wherein the organic solvent is selected from the group consisting of chloroform, methanol or mixtures thereof.

35. The process as claimed in claim 34, wherein the organic solvent is a mixture of chloroform and methanol and is added in a ratio of 2: 1.

36. The process as claimed in claim 33, wherein the buffer solution is selected from the group consisting of phosphate buffered saline (PBS) or simulated body fluid (SBF).

37. The process as claimed in claim 33, wherein the liposome suspension of step (c) is sonicated using a probe sonicator with 40 - 50 % intensity.

38. The process as claimed in claim 33, wherein chloroauric acid, liposome and ascorbic acid in step (e) are added in the ratio of 1 :2:4.

39. The process as claimed in claim 33, wherein said nano-construct of step (e) is degraded either by enzyme, increase in temperature or photo-thermal.

40. The process as claimed in claim 33, wherein said nano-construct of step (e) is transformed by lipase enzyme, ultrasound, probe sonication or defibrillation.

41. A method of treating cancer comprising administering to a subject an effective amount of degradable nano-construct as claimed in claim 1.

42. A method for prophylaxis or treatment of deep vein thrombosis comprising administering to a subject an effective amount of transformable nano-construct as claimed in claim 17.

. A stent carrying the transformable nano-construct as claimed in claim 17.