3. PRE AMBLE TO THE DESCRIPTION COMPLETE SPECIFICATION

The following specification describes the invention

The Invention relates to the Art, Method, Manner, Process and System of Preparation of Curcumin Loaded Chitin Nanogels for Skin Penetration"

FIELD OF THE INVENTION

Drug delivery through transdermal route is one of the most important modes of drug administration when oral route fails due to many reasons. Being a non-invasive route, it has advantages over injections. But skin imparts barrier functions and does not ! easily permit the entry of majority of drugs. Nanoformulations are good option to enhance penetration and nanogels have environment responsive property as well.

An example where deep skin penetration is important is Melanoma, the most serious type of skin cancer that affects the lowest layer of viable epidermis. So a formulation with good skin permeability and retention in this area would be beneficial compared to any other mode of treatment. Further, for this application, studies have shown that direct exposure to Curcumin can have significant benefits. Curcumin, however, is now a generic potential drug for other applications as well, however, because its bioavailability through the oral route is extremely low, use of curcumin has not yielded promising results by this route. Deep skin penetration of curcumin would allow curcumin to be substantially more bioavailable as it would be absorbed by the capillary network in the dermis region of skin. An example where curcumin has been explored as a drug is oral cancer for which the oral route of delivery is being explored without significant success.

The current invention presented here is an application based product development namely Curcumin loaded chitin nanogels (CCNGs), for deep skin penetration. It was prepared by a surfactant free route. These CCNGs have good size and surface properties, high stability, excellent capacity for drug loading and release and good skin penetration property. CCNGs due to its efficiency of skin penetration would be a novel approach for trafficking of curcumin through the surface layers of skin. It would have excellent application for skin cancer or other diseases where high bioavailability of curcumin through transdermal route will make the treatment effective.

BACKGROUND OF THE INVENTION

Curcumin loaded micro and nanoparticles have been well studied by various research groups for oral route of administration. However as mentioned, the most important limitation reported is the low biodistribution and bioavailability, thus hindering the effectiveness against diseases like cancer.

We have developed Curcumin loaded nanoformulation using biocompatible and biodegradable chitin, which has the capacity to form polyelectrolyte complexes, making it a very versatile material with extensive application in the biomedical and biotechnological fields. In addition, due to the rich -OH and -NHCOCH3 groups, chitin chains can sequester different ions into the gel network, another advantage for drug loading. The developed CCNGs have environment responsive property plus the benefits of nanoscale size. Both these will contribute to favour the site specific delivery of drug in the body. As the preparation does not require organic solvents or surfactants, chemical toxicity is not a concern. Chitin nanogel is an ideal candidate for skin penetration for the following reasons.

3. As the permeability of a substance through skin is inversely proportional to its size under certain conditions, the nanoscale size of CCNGs is one major factor facilitating penetration

4. The cationic charge of chitin can facilitate the penetration through intact skin

5. Chitin as well as curcumin are lipophillic in nature but the nanogel is hydrophilic, this hydrophilic-lipophillic balance of the formulation is highly beneficial

6. As a good transdermal system, beyond allowing desirable amount of drug to cross the skin barrier, the system has to be biocompatible, preferentially biodegradable and non-irritant to skin. Chitin is a very good candidate in these aspects.

SUMMARY OF THE INVENTION

In summary, novel chitin nanogel loaded with the phytodrug curcumin was prepared for the first time with significant skin penetration capacity. The CCNGs were prepared in an aqueous solution via in-situ encapsulation of curcumin into the chitin nanogel. Here, the regeneration of chitin involves precipitation in nanoscale size. This is facilitated by probe sonication at higher amplitude. The nanogel prepared by regeneration chemistry can undergo a pH induced volume phase transition. This property is very important for the therapeutic activity at the desired site by pH influenced drug release. That means the nanohydrogel swells and releases the drug at acidic pH rather than neutral or alkaline pH. This is favourable because the pH at any cancerous tissue is in the acidic range. The results obtained suggest that these CCNGs with good size and surface properties, excellent structural stability and change in physical properties in response to a pH change and good skin penetration property can be a good option for curcumin delivery via the skin portal with high bioavailability. The skin penetration properties shown in ex-vivo experiments suggest that these CCNGs may be used to attain systemic therapeutic concentration of the drug by transdermal delivery.

DESCRIPTION OF THE DRAWINGS

Fig. 1: Data showing ex-vivo skin penetration studies
Fig. 2: Fluorescent images of porcine skin sections after the skin permeation studies with control curcumin and CCNGs.

Fig. 3: Cytocompatibility of CCNGs on Human Dermal fibroblast and VERO cells respectively by MTT assay after 48 h.

Fig. 4: MTT assay for chitin nanogel (control), Curcumin (control) and CCNGs on A375 cells after 48 h exposure.

Fig. 5: Showing cellular localization of curcumin on A375 after incubation with CCNGS at different time periods by fluorescent microscopy.

Fig.: 6: Showing FACS based Apoptosis analysis for the control curcumin, chitin
nanogel and CCNGs on A375 cells after 24 h exposure.

Fig. 7: Swelling and Curcumin release behaviour of the nanogels at different pH.

Fig. 8: synthesis route for the CCNGs by controlled regeneration chemistry

Fig.9: Showing the stable CCNGs and bare chitin nanogel at pH 5.0, size distribution
of CCNGs in the SEM and DLS analysis

DETAILED DESCRIPTION OF THE INVENTION I PROOF OF INVENTION

1. Skin penetration of CCNGs

An ex-vivo experiment was carried out using full thickness porcine skin in a vertical Franz diffusion (FD) cell. The skin from the pig ear after separating the underlying cartilage and subcutaneous fat was sandwiched between the donor and acceptor compartment of the FD cell. 1-3ml of the CCNG dispersion was added in the donor
compartment on the stratum corneum side of the skin and phosphate buffer pH 4.5 was taken in the acceptor compartment, which was stirred using magnetic stirrer. 0.5- 1ml of the acceptor fluid was taken at 1h interval from 0 to 6 h and then at 24 h and replaced with equal quantity of fresh buffer. The amount of curcumin penetrated was quantified by HPLC method using C-18 column and 5% Acetic acid- acetonitrile (25: 75) as mobile phase.

The skin samples from the above experiments were punched into pieces of 1- 2cm.2The freezing liquid was added on the skin sample and allowed to freeze at -20°C. The frozen samples were sectioned using a cryotome (Leica CM 1505 S) into10-50pm sections and these cryosections were grouped into three as upper, middle and lower sections and retained Curcumin was extracted by adding 1-5ml ethanol and incubating for 12-24hr. These were then centrifuged at 5000 rpm for 15-30 min and the supernatant filtered using 22pm syringe filter. The HPLC analysis for curcumin was then carried out as mentioned above.

The transdermal flux calculated as the slope of the plot in Fig:1 for nanogels was found to be higher than that for control curcumin solution. The lag time(t lag)- symbolising the time of delay which describes the first contact of the drug with skin's surface until a steady state flux is established was found as the intercept of the plots(Fig:1). The lag time for CCNGs was found to be about half as that of curcumin solution. From the fluorescent images of skin (Fig: 2) after exposure to the nanogels and control curcumin solution, it is clear that the retention of curcumin in different layers of skin is more for CCNGs as observed by increased fluorescence.

2. cytotoxicity of CCNGS on Melanoma Vs normal cell lines

Cytotoxicity experiments were carried out on VERO (kidney epithelial cells of the African Green Monkey, NCCS Pune) and Human dermal fibroblast cells (Procell,Germany) and A375 (Human melanoma cell lines, NCCS Pune. MTT [3-(4, 5- Dimethylthiazole-2-yl)-2, 5-diphenyl tetrazolium] assay for cytotoxic evaluation is a colorimetric test based on the selective ability of viable cells to reduce the tetrazolium component of MTT in to purple colored formazan crystals. The cells were seeded on a 96 well plate with a density of 9- 10x 103cells/ cm2. After reaching 90% confluency, the cells were washed with PBS buffer and different concentration of the Curcumin loaded nanogel (100-150μl) were added and incubated. Cells in media alone devoid of nanogels acted as negative control and wells treated with Triton X-100 as positive control for a period of 24 h. The cells were incubated with 100 μl of the MTT solution for 2-4h to form formazan crystals by mitochondrial dehydrogenases. 100-150(4.1 of the solubilisation buffer (10% Triton X-100, 0.1 N HCI and IPA) was added in each well and incubated at room temperature for 1-2h to dissolve the formazan crystals. The optical density of the solution was measured at a wavelength of 570 nm using a Beckmann Coulter Elisa plate reader (BioTek Power Wave XS). Triplicate samples were analyzed for each experiment.

Cell uptake studies

Acid etched cover slips kept in 24 well plates were loaded with A 375 cells with a seeding density of more than 20000 cells per cover slip and incubated for more than 24 h for the cells to attach well.

After the incubation ,nanogel formation at a concentration of 0.5-1 mg /ml was added a long with the media in triplicate to the wells and inclubated for incubated for 4-6 h. thereafter the media with sample were removed and the cover slips were processed with Para Formaldehyde (PFA) for fluorescent microscopy. The cover slips were air dried and mounted on to glass slides with DPX as the mountant medium. The slides were then viewed under the fluorescent as well as confocal microscope to study the cell uptake. Apoptosis study Phosphatidylserine (PS) translocation from the inner to the outer layer of plasma membrane is one of the important earliest apoptotic features. The PS exposure in A375 cells was detected using an Annexin V-FITC/PI Vybrant apoptosis assay kit (Molecular probes, Eugene, OR). After reaching more than 90% confluency, the cells were treated with different concentrations of CCNGs as described previously. After being exposed to samples for more than 24 h at 38°C, cells were harvested by trypsinization and washed with PBS for 5-10 min followed by centrifugation at 500 -1000g at 4-6°C. The supernatant was discarded and the pellet resuspended in ice-cold 1X Annexin binding buffer (5x105-5x106 cells/mL). Annexin V-FITC and PI mixture were added to 100-200 pi of the cell suspension and mixed gently. The samples were then incubated at room temperature for 15-30 min in the dark. After incubation, 400-600 μL of ice-cold 1X binding buffer was added, mixed gently, and analyzed by flow cytometry. Cells in media alone devoid of nanogels (negative control) and cells treated with bare nanogels were also analyzed in the same way. Samples were analyzed in triplicates for each experiment.

The MTT assay showed that CCNGS have a specific toxicity on A375compared
to the normal Human dermal fibroblast and VERO cells as shown in the Fig.3 and Fig.4 respectively. This throws light on the fact that CCNGs can specifically treat the melanoma without serious effect on the normal cells. Fig.5 shows the internalization of CCNGs by the A 375 cells at different incubation times as observed by fluorescent microscopy.

Fig.6 shows apoptotic profile of bare chitin gel, control curcumin solution and CCNGS. It is evident from the figure that there is no significant apoptosis induced by the bare nanogels whereas the CCNGs at the higher concentration of the cytotoxic range showed comparable apoptosis as that of control curcumin. It is a well known fact that Curcumin induce apoptosis by different mechanisms and evenafter the nanogel loading, the drug retained its cytotoxic efficacy.




3. Swelling and drug release behaviour

The swelling behaviour of the nanogels was studied at different pH conditions since the product under claim is nano hydrogel which changes the physical properties in response to pH. The swelling behaviour was studied by measurement of swelling ratio at 3 different pH as acidic, neutral and basic (pH 4, 7 and 9 respectively). The dry weight (Wo) of freeze dried pelletized nanogel was noted and then placed in corresponding pH solution for sometime and then wet weight (Ww) noted after removing the surface adsorbed solution.

Swelling ratio = Ww-Wo/Wo

Drug release was studied by using pellets of the prepared drug loaded formulations. Known weight of pellets were immersed in PBS of 2 different pH values, 7.4 and 4.5 with temperature kept at 37 °C and placed in an incubator shaker at 120 -150 rpm. At predetermined intervals, aliquots of 100-200 μl were withdrawn at certain time intervals and replaced with an equal volume of fresh buffer. These samples were aliquoted into marked wells in a 96 well plate and into each well, 100-200 μl methanol was added. The concentration of the drug was monitored with a plate reader at the wavelength of 429nm. The percentage drug release was determined by the following equation

Drug release [%] = c (t) /c (0) x 100

Where c (0) and c (t) represents the amount of drug loaded and amount of drug released at a time t, respectively. All experiments were done in triplicate.

It is evident from Fig. 7 that the swelling as well as drug releases from CCNGs is pH dependent and is prominent at acidic pH than at neutral pH. This shows the pH responsive behaviour of the nanogels. The release at acidic pH is beneficial because of two reasons. One is that the pH at tumor site is acidic which favours site specific release of the drug and secondly, Curcumin under in vitro conditions show rapid degradation at and above neutral pH.

Formulation of CCNGs

Chitin solution was prepared according to literature using saturated CaCI2/methanol solution. From this solution chitin was regenerated by desolvation/precipitation by drop wise addition of methanol containing curcumin (twice the volume of the gel solution). Chitin is regenerated as nanogel entrapping curcumin as a slightly turbid solution. The above solution was then washed with water several times till the whole water got completely oozed out from the gel.


Fig.8
The gel was centrifuged with water followed by probe sonication at higher amplitude for 5-15 min, and then resuspended in water for further studies. As the amplitude and time of probing increases the particle size decreases (Table 1). The DLS data and SEM images (Fig. 9) show the nanoscale size of the formulation.

TABLE 1. Showing the particle size parameters for the CCNGs with respect to time and amplitude of probe sonication.
Fig. 9

CLAIMS

1. A method for the preparation of chitin nanogel loaded with curcumin without using any organic solvents or surfactants which show deep skin penetration and increased bioavailability of curcumin at sites of cancer with low pH.

2. A component according to claim 1, having a particle size around 100-150nm and good entrapment efficiency for the drug

3. A component according to claim 1 showing controlled curcumin release profile and increased swelling at acidic pH conditions with good hemocompatibility. This indicates that this formulation is a good candidate for cancer treatment since the pH at tumour site is acidic.

4. A component according to claim 1 having high cellular uptake by melanoma cells.

5. A component under claim 1 having potential selective toxicity towards melanoma cells (A375) in comparison to normal VERO and dermal fibroblast cells.