FIELD OF INVENTION:
The present invention relates to the field of grapheme nanocomposties for targeted, sustained and controlled release of therapeutic agents. The present invention in particular relates to hydrogel-graphene quantum dots (GQDs) nanocomposites and a method to synthesize the same for targeted, sustained and controlled release of therapeutic agents.
DESCRIPTION OF THE RELATED ART:
Graphene is a type of single layer two-dimensional nanomaterial featured with sp 2 hybridized carbon atoms arranged in a honeycomb lattice. Graphene is becoming a increasing recognized by the scientific community owing to its considerable unique properties in physical and chemical aspects. Specifically, these unique properties include high surface area, strong mechanical strength, extremely low toxicity and ease of functionalization, just to name a 12 few. The derivatives of graphene, e.g. graphene oxide (GO) and GQDs inherits the majority of the superior properties of graphene, and they have been broadly investigated for a diversity of applications. The article entitled “Graphene quantum dots-based drug delivery for ovarian cancer therapy” talks about graphene quantum dots (GQDs) based nano-sized drug delivery systems (DDS) for ovarian cancer treatment. As a starting point, the facile synthesis method of the GQDs was established. Subsequently, the targeting ligand,folic acid (FA), was conjugated to GQDs. Next, a FDA approved chemotherapeutic drug, Doxorubicin (DOX), was loaded to form the GQDs-FA-DOX nano-conjugation as the DDS. Moreover, the uptake profile and anti-cancer effect of the GQDs-FA-DOX were validated in ovarian cancer cells. Finally, the immunotoxicity of GQDs and its mechanism were investigated and elucidated [Yiru Qin; scholarcommons, April 27, 2016]. The article entitled “Sulfur-doped graphene quantum dots as a novel fluorescent probe for highly selective and sensitive detection of Fe3+” talks about the sulfurdoped graphene quantum dots (S-GQDs) with stable blue-green fluorescence were synthesized by one-step electrolysis of graphite in sodium ptoluenesulfonate aqueous solution. Compared with GQDs, the S-GQDs drastically improved the electronic properties and surface chemical reactivities, which exhibited a sensitive response to Fe3+. Therefore, the S-GQDs were used as an efficient fluorescent probe for highly selective detection of Fe3+. Upon increasing
- 3 -
of Fe3+ concentration ranging from 0.01 to 0.70 µM, the fluorescence intensity of S-GQDs gradually decreased and reached a plateau at 0.90 µM. The difference in the fluorescence intensity of S-GQDs before and after adding Fe3+ was proportional to the concentration of Fe3+, and the calibration curve displayed linear regions over the range of 0–0.70 µM [Shuhua Li, Yunchao Li, Jun Cao, Jia Zhu, Louzhen Fan, and Xiaohong Li; Anal. Chem., 2014, 86 (20), pp 10201– 10207, 2014].
The article entitled “pH-sensitive dimethylaminoethyl methacrylate (DMAEMA)/acrylamide (AAm) hydrogels: Synthesis and adsorption from uranyl acetate solutions” talks about the DMAEMA content of the gel, the pH, and the ionic strength of the solution decreased the swelling ratios of the hydrogels. The adsorption of poly(DMAEMA/AAm) hydrogels from uranyl acetate (UA) solutions was studied at different pHs. The adsorption capacity of hydrogels increased from 200 to 1200 mg of UA per gram of dry hydrogel with increasing pH of the adsorption solution [Berna Yildiz, Belma Isik, Mehmet Kis, Özgen Birgül; Inc. J Appl Polym Sci 88: 2028–2031, 06 March 2003].
However, different type of cancers treatment requires different drugs which alter with the drug carriers. Prior arts focus on ovarian cancer.
IN Publication No. 876/MUMNP/2012 relates to a cellulose aerogel comprises a plurality of cellulose nanoparticles. The cellulose nanoparticles preferably comprise at least 50% or 80% cellulose nanocrystals by weight of cellulose nanoparticles, and the cellulose nanoparticle aerogel preferably has a density of from 0.001 to 0.2g/cm3 or from 0.2 to 1.59g/cm3 The cellulose nanoparticle aerogel typically has an average pore diameter of less than 100 nmm and the cellulose nanoparticles may comprise anionic and/or cationic surface groups. However, this procedure requires several solvent exchange steps, making the entire synthesis procedure very complex. The incorporation of carbon nanotubes may compromise the biocompatibility of their aerogels. Moreover, theirs is not a smart (stimuli – responsive material). They are using cellulose as monomer which fall under the category of natural monomers.
- 4 -
IN Publication No. 3244/DEL/2013 (EP3063091 (A1)) relates to GQDs, Size and ROS to reduce their cytotoxicity. Small size can damage cell organelles and production of ROS- (reactive oxygen species) can ‘hamper cell machinery in multiple ways. We have shown that cytotoxicity can be significantly reduced by embedding GQDS inside the PEG matrix rather than creating a thin shell around each GOD. However, the GQDs they synthesized were cytotoxic in nature and lead to the production of reactive oxygen species (ROS). The cell viability as seen from their MTT assay results was only 60%. They have used a homopolymer matrix of a natural polymer for embedding their GQDs.
IN Publication No. 201637033050 relates to a DOPA melanin (DM) as well as compositions comprising the DM polymer and uses thereof. The method comprises contacting a reactant such as 3 4 dihydroxyphenylalanine (DOPA) with a high concentration aqueous salt solution under oxidative conditions. The resulting DM polymer may form as insoluble particles or as a coating on a substrate surface. However, the polymer matrix has a negative surface charge due to which they can encapsulate only cationic drugs.
Publication No. US2015069295 (A1) relates to a hydrogel composition for photocatalytic hydrogen production and storage. The composition containing a graphene, a TiO2 nanotube array, and a carbon quantum dot defines a threedimensional porous and continuous cross-linked structure. Also disclosed is a method of producing this composition. However, the composites made with carbon quantum dots are being used for the photocatalytic production of hydrogen. They did not report any biomedical applications. Moreover, metal oxide nanotubes and carbon quantum dots are known to induce Cytotoxicity.
Hence, the present invention aims to provide hydrogel-graphene quantum dots (GQDs) nanocomposites and a method to synthesize the same for targeted, sustained and controlled release of therapeutic agents.
OBJECTS OF THE INVENTION: The principal object of the present invention is to provide hydrogel-graphene quantum dots (GQDs) nanocomposites and a method to synthesize the same for targeted, sustained and controlled release of therapeutic agents.
- 5 -
Another object of the present invention is to provide physically crosslinked, biocompatible “GQD-hydrogel nanocomposites”, not restricted to neat GQDs as in prior arts for targeted, sustained and controlled release of various therapeutic agents in response to pH.
Yet another object of the present invention is to provide the hydrogel nanocomposite as a suitable drug carrier owing to their ability for pH sensitive drug delivery of a biologically active agent for site-specific and sustained drug delivery.
Still another object of the present invention is to synthesize graphene quantum dots (GQDs) from agrowaste that are highly biocompatible.
SUMMARY OF THE INVENTION: The present invention discloses hydrogel-graphene quantum dots (GQDs) nanocomposites and a method to synthesize the same for targeted, sustained and controlled release of therapeutic agents. Hydrogel nanocomposite is prepared via scalable, single-step free radical aqueous co-polymerization of Acrylic acid (AAc) and 2-(Dimethylamino)ethylmethacrylate (DMAEMA) with filler concentration ranging from 0.01% - 0.3% weight/volume without the use of any chemical crosslinkers. These PAD-GQD nanocomposites displayed better swelling and release characteristics. This invention provides the hydrogel nanocomposite as a suitable drug carrier owing to their ability for pH sensitive drug delivery of a biologically active agent for site-specific and sustained drug delivery.
Present invention provides poly(AAc-co-DMAEMA) hydrogel matrix using synthetic monomers wherein graphene quantum dots are embedded, producing fluorescent hydrogel nanocomposites which are physically crosslinked and pH responsive smart fluorescent composite materials synthesized via one step free radical aqueous copolymerization. These hydrogel composites show more than 4000% swelling with an almost 86% release of the encapsulated drugs.
The GQDs of the present invention are synthesized from agrowaste and are highly biocompatible showing significant cell viability with DF-1 (92%), HEK293
- 6 -
(95%) and HepG2 (88%) as reported in paper titled "Sustainable synthesis of single crystalline graphene quantum dots for bioimaging and beyond” published in Green Chemistry. (Sujata Sangam*, Apoorv Gupta*, Adeeba Shakeel, Rohan Bhattacharya, Arun Kumar Sharma, Deepa Suhag, Sandip Chakrabarti, Sandeep Kumar Garg, Sourav Chattopadhyay, Biswarup Basu, Vinod Kumar, Satyendra Kumar Rajput, Malay Kishore Dutta and Monalisa Mukherjee; Green Chem., 2018,20, 4245-4259. DOI- 10.1039/C8GC01638K)
However, cell viability and compatibility is not compromised, rather increased, after the composite formation. The GQD-nanocomposite hydrogels showed no significant differences in the cell viability and cell morphology. The entire biocompatibility assay is done where they are tested for both in-vitro through MTT assay and in-vivo compatibility through Gamma scintigraphy. Further, the composites are prepared via simple one-step free radical aqueous copolymerization of Acrylic acid and Dimethyl aminoethyl methacrylate alongwith GQDs as nanofillers which is facile and economic.
Present invention provides encapsulation of any drug irrespective of its surface charge and composition. It can easily encapsulate synthetic, herbal and even protein drugs without any damage, loss of activity or degradation of the active agent. It is an established fact that chemical crosslinkers, present even in small proportions, can impart toxicity to the material and needs to be removed prior to application. In order to avoid this in the present invention in-situ physical crosslinking technique is generated for the synthesis of hydrogels nanocomposites.
BREIF DESCRIPTION OF THE DRAWINGS: It is to be noted, however, that the appended drawings illustrate only typical embodiments of this invention and are therefore not to be considered for limiting of its scope, for the invention may admit to other equally effective embodiments. Figure 1 shows scheme 1 Synthesis of PAD-GQD hydrogel nanocomposites; Figure 1a shows ATR-FTIR spectra of PAD, PAD-GQD 0.05% and PAD-GQD 0.3% Figure 2 shows Equilibrium swelling study;
- 7 -
Figure 3 shows a) SEM and b)TEM micrographs of swollen PAD hydrogel nanocomposite; Figure 4 shows XRD of PAD, PAD-GQD 0.05% and PAD 0.3% Figure 5 shows Cumulative drug release profile; Figure 6 shows Rheology of PAD, PAD-GQD 0.05% and PAD-GQD 0.3% Figure 7 shows Thermogravemetric analysis of PAD, PAD-GQD 0.05% and PAD-GQD 0.3% Figure 8 shows Cytotoxic assessment of PAD, PAD-GQD 0.05% and PAD-GQD 0.3% Figure 9 shows Histological assessment of rat vital organs after 15 consecutive days of oral administration of PAD, PAD-GQD 0.05% and PAD-GQD 0.3% Figure 10 shows Gamma Scintigraphy scans of rabbit after oral administration of PAD, PAD-GQD 0.05% and PAD-GQD 0.3%
DETAILED DESCRIPTION OF THE INVENTION: The present invention provides a discloses hydrogel-graphene quantum dots (GQDs) nanocomposites and a method to synthesize the same for targeted, sustained and controlled release of therapeutic agents. Hydrogel nanocomposite is prepared via scalable, single-step free radical aqueous co-polymerization of Acrylic acid (AAc) and 2-(Dimethylamino)ethylmethacrylate (DMAEMA) with filler concentration ranging from 0.01% - 0.3% weight/volume without the use of any chemical crosslinkers. These PAD-GQD nanocomposites displayed better swelling and release characteristics. This invention provides the hydrogel nanocomposite as a suitable drug carrier owing to their ability for pH sensitive drug delivery of a biologically active agent for site-specific and sustained drug delivery.
Present invention provides poly(AAc-co-DMAEMA) hydrogel matrix using synthetic monomers wherein graphene quantum dots are embedded, producing fluorescent hydrogel nanocomposites which are physically crosslinked and pH responsive smart fluorescent composite materials synthesized via one step free radical aqueous copolymerization. These hydrogel composites show more than 4000% swelling with an almost 86% release of the encapsulated drugs.
- 8 -
The present invention relates to development of sulphur doped 0D graphene quantum dots (GQDs) (filler) hydrogel nanocomposite with varying filler % via a facile, scalable and cost effective technique of single step aqueous free radical copolymerization of AAc and DMAEMA in GQDs aqueous solution to develop a series of physically cross-linked, stable, superabsorbent and pH-responsive hydrogels. Characterization of these poly(AAc-co-DMAEMA)-Graphene quantum dots (PAD-GQD) hydrogel nanocomposites matrices is established in terms of chemical composition, glass transition, mechanical properties, morphology and pH-dependent swelling and release property and is represented by virtue of the various examples presented hereinafter and is in no way limiting. Additionally, preliminary cytotoxic effects of the hydrogel nanocomposites are also examined in human embryonic kidney cells. Thereby, this further validates the fact that these hydrogels are cytocompatible and can be potentially used for oral delivery of drugs. Biocompatibility assay which comprises of histological and hemolysis analysis has also been performed which has shown positive results with hydrogel nanocomposites with different filler percentage. The in-vivo drug delivery studies has been carried out on rabbit models with hydrogel nanocomposites loaded with therapeutic agents as well as the hydrogel nanocomposite and monitored by Gamma Scintigraphy showing targeted, sustained and controlled release of the drug.
The GQDs of the present invention are synthesized from agrowaste and are highly biocompatible showing significant cell viability with DF-1 (92%), HEK293 (95%) and HepG2 (88%) as reported in paper titled "Sustainable synthesis of single crystalline graphene quantum dots for bioimaging and beyond” published in Green Chemistry.
However, cell viability and compatibility is not compromised, rather increased, after the composite formation. The GQD-nanocomposite hydrogels showed no significant differences in the cell viability and cell morphology. The entire biocompatibility assay is done where they are tested for both in-vitro through MTT assay and in-vivo compatibility through Gamma scintigraphy. Further, the composites are prepared via simple one-step free radical aqueous copolymerization of Acrylic acid and Dimethyl aminoethyl methacrylate alongwith GQDs as nanofillers which is facile and economic.
- 9 -
Present invention provides encapsulation of any drug irrespective of its surface charge and composition. It can easily encapsulate synthetic, herbal and even protein drugs without any damage, loss of activity or degradation of the active agent. It is an established fact that chemical crosslinkers, present even in small proportions, can impart toxicity to the material and needs to be removed prior to application. In order to avoid this in the present invention in-situ physical crosslinking technique is generated for the synthesis of hydrogels nanocomposites.
The invention is described in detail with reference to the examples given below. The examples are provided just to illustrate the invention and therefore, should not be construed to limit the scope of the invention. EXPERIMENTS Experiment 1 Method for preparation of hydrogel nanocomposite. Ex-situ synthetic method was adopted for the nanocomposite formation. PADGQD hydrogel nanocomposites were synthesized by aqueous free-radical polymerization using AAc, DMAEMA, GQDs, SPS and TEMED, as shown in Scheme 1. Table I shows feed composition of PADP Hydrogels. The GQDs water dispersion with various concentrations (ranging from 0.05 to 0.3 weight percent) were used for the synthesis. Briefly, each hydrogel was prepared by first mixing AAc and DMAEMA at ~ 0 °C (ice-bath) in a two-necked round bottom flask for about 5 min. GQDs water solution, concentrated aqueous solution of SPS (0.025 mol% of monomers) and TEMED (0.3 mol% of monomers) were then added to the monomer mixture and mixed with constant nitrogen purging for about 10 minutes.The reaction mixture was then sonicated for 2 minutes. The prepared reaction mixture was then poured in cylindrical poly (vinyl chloride) (PVC) moulds and incubated for 24 h at 41 ± 1 °C. The synthesized PAD-GQD hydrogel nanocomposites thus prepared were removed from the moulds after the reaction. Unreacted water-soluble constituents were removed from the hydrogel pieces by washing them in distilled water for five consecutive days. The gels were dried to a constant weight by drying in air for 24 h, followed by drying in vacuum oven for 5 h at 40 °C.
- 10 -
Table 1 :
Note: APS = 0.025 mole % and TEMED = 0.3 mole %; *mol % to total monomer content
Experiment 2 FTIR measurement The FTIR measurements were done on an ATR-FTIR model Alpha-P, Bruker of dried powdered PAD-GQD hydrogel nanocomposites. The IR spectrum of each hydrogel was obtained by scanning at a resolution of 4cm-1 and by averaging 24 scans. The spectra of PAD, PAD-GQD 0.05% and PAD-GQD0.3% shows a broad absorption band centering at ~ 3497 cm-1, ~3490 cm-1 and 3431 cm-1 respectively arising either due to the ?OH (O-H Stretch) of carboxylic acids or due to moisture. However, presence of moisture in hydrogel nanocomposite samples is unlikely as the samples were thoroughly dried and kept in desiccator till use, thus, indicating the presence of PAAc units in the copolymer. Rise in the intensity of these bands for PAD-GQD nanocomposites as compared to only PAD shows the presence of –COOH/O—H groups of GQDs in addition to AAc units, thereby, indicating successful inclusion of GQDs in PAD matrices. The copolymer and the nanocomposites show a single peak in the region 1740-1732 cm-1, which may be due to carbonyl stretch of PDMAEMA and not PAAc. This is because the carbonyl groups of PAAc have been converted into carboxylate
- 11 -
anions due to the presence of tertiary amine groups of PDMAEMA. The carboxylate ions thus formed show a strong symmetrical stretching band near 1650-1550cm-1. Possibly, in the as-prepared hydrogel samples all the carbonyl groups of PAAc are in form of carboxylate anion (-COO-). This is also an affirmation that the PADP copolymer contains both carboxylic acid (PAAc) and tertiary amine (PDMAEMA). This reaffirms the electrolytic complexation between the two. The appearance of the bands in the range of ~3410-3500 cm-1 indicates the presence of both N-H and –OH groups confirming the formation of the copolymer of AAc and DMAEMA in both the copolymer and hydrogel nanocomposite samples. In addition, peak at ~2358 cm-1 signifies sp3 C-H stretching vibrations in all the three samples. This peak is intensified in both PAD-GQD 0.05% and PAD-GQD 0.3% nanocomposites signifying the presence of GQDs in the polymer matrix. Increase in the intensities of –OH peaks in the PAD-GQD samples, shows successful hydrogen bonding between the GQDs and the polymer matrix. The peaks are intensified as the percentage of GQDs as filler is increased in hydrogel nanocomposites. The appearance of peak at ~780 cm-1 in PAD-GQD nanocomposite samples originates due to the C-S stretching, further proving the incorporation of sulphur doped GQDs in the polymer matrix.
Experiment 3 Swelling Study The swelling studies were carried out PBS buffer solution of pH 7.4 at 37 ± 1 °C. Ionic strength of all the buffer solutions was kept roughly constant by adding 1 M KCl salt. The initial weights (M0) of dry hydrogel and hydrogel nanocomposite samples were noted and finally submerged in buffer solutions. The gels were taken out after every predetermined time period and their swollen weight (Mt) was noted. A moist filter paper was used to soak the surface water of the swollen gels before weighing. The swelling study was performed until the gels reached their equilibrium weight. The swelling ratio (Qt), at any particular time (t), was calculated by using the formula: Qt= (Mt - M0)/M0 X 100 (1) The study of swelling mechanism is an important factor influencing the diffusive delivery of drugs and the ability of the hydrogel to be used as a drug delivery system. The polyelectrolytic hydrogel and its nanocomposites were established to have a pH-dependent swelling behavior due to the presence of amphiphilic
- 12 -
ionizable pendant groups present in the DMAEMA and AAc units. At pH ~3.5 (isoelectric point, pI) minimum swelling in the hydrogel nanocomposite was observed, which is a characteristic behaviour of polyampholytes. When immersed in the PBS buffer, it was seen that the hydrogels showed rapid swelling rate in the first 10 hours and attained equilibrium after 24 hours. As the pH is increased or decreased, the interpolymer complexes dissociate due to ionization of the pendant groups and the electrostatic repulsion between them results in the swelling and deswelling of the hydrogel matrices. Swelling of hydrogel nanocomposite at pH 7.4 ionization (deprotonation) of the AAc units (-COO) accounts for the swelling at higher pH. Equilibrium swelling for different samples increased in the order of PAD