Description:Form 2

THE PATENT ACT, 1970
(39 of 1970)
&
THE PATENT RULES, 2003
COMPLETE SPECIFICATION
(See section 10 and rule 13)

“GRAPHENE OXIDE-CHITOSAN BASED SCAFFOLDS AS A POTENTIAL DRUG DELIVERY CARRIER”

in the name of UNIVERSITY OF MADRAS an Indian National having address at UNIVERSITY OF MADRAS, CHEPAUK, CHENNAI, CHENNAI – 600005, TAMIL NADU, INDIA.

The following specification particularly describes the invention and the manner in which it is to be performed.

SOURCE AND GEOGRAPHICAL ORIGIN OF THE BIOLOGICAL MATERIAL:

SL.
NO COMMON NAME SCIENTIFIC NAME PART OF BIOLOGICAL SOURCES SOURCE OF ACCESS DETAILS OF GEOGRAPHICAL LOCATION
1. Pink shrimp Pandalus borealis Others- Chitosan obtained from shrimp Shell Trader (i) Name of the trader :
Sigma-Aldrich Chemical Pvt Limited
(ii) Contact Details:
Industrial Area, Anekal Taluka, Plot No 12,12 Bommasandra - Jigani Link Road-, Bangalore-560100, India

FIELD OF THE INVENTION:

The present invention generally relates to drug loaded with effective delivery carriers. More particularly, the present invention relates to process of preparation of drug loaded graphene oxide-chitosan based scaffold for controlled released of drug and product thereof.

BACKGROUND OF THE INVENTION:

A variety of methods are known to formulate pharmaceutical compositions to provide various release patterns of the active agent from the composition. However, research continues in an effort to develop improved compositions capable of delivering pharmaceutical agents in a sustained and predictable manner.

The advantages of sustained release formulations are well known in the pharmaceutical field. These include the ability of the given pharmaceutical preparation to maintain a desired therapeutic effect over a comparatively longer period of time, reduced side effects, etc. Moreover, for drugs having a short elimination half-life, less frequent administration and better patient compliance may be obtained with sustained release preparations as compared to the conventional dosage forms.

Cefotaxime is a broad spectrum third generation cephalosporin and one of the most important parenterally applied antibiotics. It is generally administered in the form of its sodium salt.

There have been various patents available in the patent literature in relation to Cefotaxime. Some of them are mentioned below.

WO1996020198A1discloses a process for the production of cefotaxime in acetone/water and its use in the production of a sodium salt of cefotaxime and a crystalline sodium salt of cefotaxime in form of rounded agglomerates and in form of needles.

WO2004063203A1 discloses improved process for the preparation of the sterile cefotaxime sodium of formula (I), from the compound of formula (II).
Susi Kusumaningrum et. al reported on efficient procedure for synthesis of cefotaxime sodium using sodium-2-ethylhexanoate as the source of sodium ion. Cefotaxime sodium is an antibiotic that has high economic value as well as the necessary but expensive. Research aims to find an efficient procedure for the synthesis of cefotaxime sodium. In this research, cefotaxime sodium was synthesized from cefotaxime acid and sodium-2-ethyl hexanoate. The optimal conditions for the synthesis were obtained as following: mole cefotaxime acid/mole sodium-2-ethyl hexanoate= 2:3; ethanol food grade as a solvent, reaction time at 10 minutes and room temperature. The yield of cefotaxime sodium was 97.11% based on cefotaxime acid and the purity was 99.10%. The chemical structure and molecular weights of products were identified with FTIR, NMR, and LC – Mass Spectrometry. The quality product testing is also done with the parameters solubility product in aqueous and acid media, absorbance value, pH in aqueous media, and purity. The products meet the requirements of the United States Pharmacopeia, Japan Pharmacopeia, and British Pharmacopeia.

Despite widespread process, no prior art discloses a potential drug delivery carrier for sustained release of Cefotaxime drug which subsequently refrain from frequent administration of Cefotaxime drug.

Hence an attempt has been made to develop a process of preparation of Cefotaxime Monohydrate drug loaded graphene oxide-chitosan based scaffolds for sustained release of drug Cefotaxime Monohydrate.

OBJECT OF THE INVENTION:

The main object of the present invention is to develop a process for preparing drug loaded scaffolds for sustained drug release.

Another object of the present invention is to develop a process for preparation of Cefotaxime Monohydrate drug loaded graphene oxide-chitosan based scaffolds for sustained release of drug Cefotaxime Monohydrate at determined rate.

Yet another object of the present invention is to evaluate drug loading and drug release efficiency of the prepared scaffolds.

Further object of the present invention is to utilize the prepared Cefotaxime Monohydrate drug loaded graphene oxide-chitosan based scaffolds for releasing Cefotaxime Monohydrate drug at a controlled rate for a prolonged period of time.

BRIEF DESCRIPTION OF THE DRAWINGS:

Figure 1 depicts prepared scaffolds.
(a) Chitosan/Graphene Oxide (10 mg) (CS/GO-1)
(b) Chitosan/Graphene Oxide (20 mg) (CS/GO-2)
(c) Chitosan/Graphene Oxide (30 mg) (CS/GO-3)

Figure 2 depicts thermo-gravimetric analysis (TGA) curve of scaffold-chitosan with different concentration of graphene oxide (a) CS GO(10 mg)-1, (b) CS/GO(20 mg)-2, (c) CS/GO(30 mg) -3 scaffolds.

Figure 3 depicts mechanism of formation of scaffold of the present invention.

Figure 4 depicts Calibration graph of drug solution used in the drug loading process.

Figure 5 depicts Drug loading mechanism of the prepared scaffolds.

Figure 6 depicts UV Visible spectrum of drug loading graphs taken at various time intervals.
(a) CS/GO-1
(b) CS/GO-2
(c) CS/GO-3
(d) Drug loading % of the prepared scaffolds with chitosan and different ratios of graphene oxide

Figure 7 depicts schematic representation of drug loading and release of the prepared scaffold.

Figure 8 depicts the cumulative drug release % of the prepared scaffolds with chitosan and different ratios of graphene oxide.

Figure 9 depicts drug release kinetics curves of prepared scaffolds (a) Zero order, (b) First order, (c) Koresmeyer-peppas model and (d) Higuchi model.

Figure 10 depicts XRD pattern of the prepared scaffolds.
(a) CS/GO-1
(b) CS/GO-2
(c) CS/GO-3

SUMMARY OF THE INVENTION:

The present invention discloses a process of preparation of Cefotaxime Monohydrate drug loaded graphene oxide-chitosan based scaffolds for sustained release of drug Cefotaxime Monohydrate at determined rate. The process of the present invention comprises of
dissolving chitosan in CH3COOH and sonicating followed by stirring to form chitosan solution;
dispersing graphene oxide in ultrapure water and sonicating to form graphene oxide solution;
mixing the chitosan solution and the graphene oxide solution under constant stirring followed by stirring and adding ß-sodium glycerol phosphate to form uniform gel network;
transferring the uniform gel network into 48 well plates and freezing followed by lyophilizing to form graphene oxide-chitosan based scaffolds;
adding aqueous Cefotaxime Monohydrate drug solution to the graphene oxide-chitosan based scaffolds and placing in an orbital shaker to form drug loaded scaffolds;
removing the drug loaded scaffolds from the drug solution and lyophilizing to form Cefotaxime Monohydrate drug loaded graphene oxide-chitosan based scaffolds.
The present invention also discloses a Cefotaxime Monohydrate drug loaded graphene oxide-chitosan based scaffolds for sustained release of drug Cefotaxime Monohydrate at determined rate prepared by the process described above.

DETAILED DESCRIPTION OF THE INVENTION:

The present invention discloses a process of preparation of Cefotaxime Monohydrate drug loaded graphene oxide-chitosan based scaffolds for sustained release of drug Cefotaxime Monohydrate at determined rate and product thereof.

The process of the present invention comprises of dissolving chitosan in CH3COOH and sonicating followed by stirring to form chitosan solution. Dispersing graphene oxide in ultrapure water and sonicating to form graphene oxide solution. Mixing the chitosan solution and the graphene oxide solution under constant stirring followed by stirring and adding ß-sodium glycerol phosphate to form uniform gel network. Transferring the uniform gel network into 48 well plates and freezing followed by lyophilizing to form graphene oxide-chitosan based scaffolds. Adding aqueous Cefotaxime Monohydrate drug solution to the graphene oxide-chitosan based scaffolds and placing in an orbital shaker to form drug loaded scaffolds. Removing the drug loaded scaffolds from the drug solution and lyophilizing to form Cefotaxime Monohydrate drug loaded graphene oxide-chitosan based scaffolds (Figure 1).

The prepared scaffold was then subjected to evaluate its thermal behavior and drug loading efficiency.

Thermal behavior of the prepared scaffolds was represented in the figure 2. A difference in weight loss of 8.3% has been observed at the temperature range of 50 to 110°C which directly corresponds to the removal of residual solvent and weak hydrogen bonds from the scaffold. The water molecules present in the interlayer of graphene oxide sheets were removed at the temperature range of 50 to 130°C. The next degradation starts from 130 to 300°C in which nearly 46% of the mass loss occurs which is mainly due to the de-polymerization and de-amination of chitosan along with the loss of -COOH, -OH and epoxy groups present in the graphene oxide. During this phase, there is a possibility of forming thermally stable NH3+ ions in the chitosan and -COOH groups present in the graphene oxide. With these interactions, the strength of chitosan increases, which drastically decreases the crystallinity as confirmed by the XRD pattern of the prepared scaffolds (figure 10). The remaining weight loss has occurred due to the polysaccharide unit’s decomposition and also the removal of stable oxygenated functional groups present in the scaffold. With increase in the concentration of graphene oxide from 10 mg to 30 mg, there is significant decrease in weight loss from 34.5% to 16.6 %. This indicates the thermal resistivity of graphene oxide which occurred mainly due to the increase in -OH moiety and also the interaction between hydroxyl and epoxide groups.

Figure 3 depicts the schematic representation of mechanism taking place during the formation of scaffolds. Chitosan carries a positive charge in amine groups which indicates the solubility of chitosan in acidic conditions. Obtaining a homogenous solution by adding dissolved state of chitosan along with graphene oxide to a suitable cross-linker ß-sodium glycerol phosphate. This cross-linker forms a hydration layer among the chitosan which induces solubility in water at neutral pH and at lower temperatures. The higher concentration of cross-linker and an increase in gelation time directly influences the mechanical property of material. This complete mechanism takes place at room temperature. The protonated amine groups present in chitosan forms an ionic interaction with the phosphate groups of the cross-linker. At the same time, the -COOH groups present in the graphene oxide forms an ionic interaction with one of the oxygen present in the phosphate group of the cross-linker. This induces a linkage between the graphene oxide, chitosan and the ß-sodium glycerol phosphate results in a formation of stable scaffold at room temperature.

Drug loading efficiency studies of the prepared graphene oxide-chitosan based scaffolds:
A series of Cefotaxime Monohydrate drug solutions were prepared by using ultrapure water with 4, 8, 12, 16, 20 µg/mL concentrations. The slope and intercept values were calculated accordingly. Figure 4 depict the linear relationship obtained from 4 µg/mL to 20 µg/mL drug solutions at ? max 235 nm. The R2 value was found to be 0.9995 indicating the linearity of the equation. The drug loading into the scaffolds is primarily based on the swelling property in aqueous medium. Since loading of drug has taken place after the preparation of scaffold, there is a better chance of swelling controlled drug loading to occur. The presence of carboxylic acid group in graphene oxide intimates the swelling at the pH greater than 7 which allows the drug to enter into the scaffold.

The drug loading mechanism has been shown in the figure 5. The -OH groups of the graphene oxide and the chitosan groups makes intermolecular hydrogen bonding whereas carboxylic acid groups present in graphene oxide makes an ionic interaction with phosphate group and primary amine group present in the cross-linker. There is also a possibility of intramolecular hydrogen bonding formation with chitosan molecules. The active functional groups present in Cefotaxime drug are NaO and the primary amine group interacts with the carboxylic acid groups of graphene oxide which makes way for formation of amide bond and also NaO forms an ionic bond with primary amine group of the chitosan.
The increase in concentration of graphene oxide increases drug loading performance of the prepared scaffolds with the help of p-p interaction between graphene oxide and drug molecules. This is mainly occurred due to the electrostatic interactions and hydrogen bonding taking place between cationic drug and functional groups present the graphene oxide structure. It is evident that higher concentration of GO enhances drug loading performance of the prepared scaffolds.
The drug loading was calculated with periodic measurements at various time intervals 0, 6, 12, 24, 48 h by using a UV-Visible Spectrophotometer were shown in the figure 6. The scaffolds were freeze dried after loading and then released in PBS medium at different time intervals 0, 2, 4, 6, 8, 10, 12, 24, 48, 72, 96 and 120 h. With increase in concentration of graphene oxide, drug loading of scaffolds increased from 44 to 58 % which indicates that more interaction occurs between the scaffold and drug molecule.

The schematic representation of the prepared scaffolds is shown in Figure 7. Initially, drug has been loaded by immersing the prepared scaffolds into drug solution for 48 h. Then, the scaffolds have been lyophilized by using a freeze dryer at -80°C for 48 h. The yellow spots represent drug molecules which have been loaded into the scaffolds. The drug loaded scaffolds has been released using a PBS solution at the pH 7.4.
Initially after 2h, the surface absorbed drugs have been released into the releasing medium which is termed as burst release. After 24 h, considerable amount of drug has been released into releasing medium. Breaking of ionic bonds formed between graphene oxide and chitosan molecule occurs resulting in the release of more amount of drug into the solution. After the observation of scaffolds in the releasing medium for 120h, a considerable amount of drug is still left over at the surface of scaffolds which indicates that the formed amide bond takes certain amount of time to degrade eventually small amount of drug has been left over since 100 % release of drug has not been achieved.
The drug solution with 20 µg/mL has been used in the drug loading process. About 50 mL of the drug-loaded solution has been taken in a 100 mL graduated reagent bottle. The calculated amount of the scaffolds has been taken for drug loading and placed on the orbital shaker rotated at 60 RPM. 3 mL of the drug solution has been pipetted out using a 5 mL micropipette and its absorbance has been measured using a UV-Visible spectrophotometer from 210 to 320 nm range. After the measurement, the solution has been added again into the drug solution. The absorbance values were noted down at 0, 6, 12, 24, and 48 h. The loading efficiency of the sample was calculated by using the formula given below
Loading efficiency (%)=(Initial concentration -concentration at a time "t")/(Initial concentration)× 100
After 48 h, the drug-loaded scaffolds have been carefully removed from the drug solution, lyophilized at -80°C for 48 h and further drug release studies were carried out.
The prepared Cefotaxime Monohydrate drug loaded graphene oxide-chitosan based scaffolds was then subjected to experimental studies to evaluate its Thermal behavior and drug release properties.
Drug Release:
The invitro drug release studies were evaluated by fitting the obtained value into four different kinetic models to predict the mechanism of the drug release. The models were (a) Zero order, (b) First order, (c) Korsmeyer-Peppas model, (d) Higuchi square root model.
The freeze-dried scaffolds after drug loading have been placed in the
60 mL PBS (pH 7.4) using a 100 mL graduated reagent bottle in the orbital shaker rotated at 60 RPM. Immediately, about 3 mL of the sample has been pipetted out using 5 mL micropipette and analyzed using a UV-Visible spectrophotometer. Simultaneously, 3 mL of the fresh PBS solution has been replaced in the drug-releasing solution. The same process has been repeated for different time intervals at 0, 2, 4, 6, 8, 10, 12, 24, 48, 72, 96, and 120 h. In order to replicate the reproducibility, triplicate experiments were performed. The cumulative drug release (%) has been calculated by using the formula mentioned below
Drug Release (%)= (Total drug loaded- drug released at a time “t”)/(total drug loaded)×100
The cumulative drug release (%) has been plotted against various time intervals.
The drug release is shown in Figure 8. After 12 h, it was found to be 62, 38, and 71% for the three scaffolds respectively. After 120 h, nearly 84, 77, 96 % of the loaded drugs were released into the solution. In continuation with the swelling property of the scaffold in the PBS medium, the drugs release into the releasing medium occurred at a controlled rate which was evidenced from the cumulative drug release graphs.

Drug release kinetics curves of the prepared scaffolds of the present invention:

The solubility, diffusion coefficient and water penetration ability determines the releasing rate of drug into releasing medium. Figure 9 shows drug release kinetics of the prepared scaffolds. The dotted points indicate the original data and the straight line indicates the fitted data. The data obtained from the different release models were evaluated by using linear regression method.

At the zero-order kinetics, graph was plotted for cumulative drug release % against drug release time intervals and found to be lower for (CS/GO-1, R2 = 0.6345) and (CS/GO-3, R2 = 0.6630). But the concentration of CS/GO -2 scaffold was found to be ideal for the zero-order kinetics release with R2 value of 0.9530. Table 1 represents drug release kinetic models along with the parameters (R2, n) for the prepared scaffolds.

Table 1: Drug release kinetic models and its parameters for the prepared scaffolds

Drug release kinetic models Parameter CS/GO-1 CS/GO-2 CS/GO-3
Zero Order R2 0.6345 0.9530 0.6630
First Order R2 0.8431 0.9895 0.9182
Higuchi R2 0.8403 0.9274 0.8674
Koresmeyer-Peppas R2
n 0.8232
0.3060 0.9818
0.5622 0.8275
0.2711

First order kinetics mainly depends on the concentration of the drug, i.e higher the concentration faster drug release rate. A graph has been plotted for drug release time intervals against the log drug remaining % and obtained a similar R2 values such as CS/GO-1 (R2 = 0.8431), CS/GO-3 (R2 = 0.9182) and found a better release for the CS/GO-2 concentration with the R2 value of 0.9895 for the scaffolds. The graph of Higuchi release model was plotted for cumulative drug release % against the SQRT of time, similar trend was followed for this model as the CS/GO-2 scaffold has the highest R2 value of 0.9274. The most prominent release model for release of drug from gel system across the releasing medium was found to be koresmeyer-peppas model which was evidenced by the graph plotted between the log of drug release (%) against log time. Further, the drug release mechanisms were evaluated by fitting with the koresmeyer-peppas model and obtained n value of 0.3060, 0.5622 and 0.2711 for the CS/GO-1, CS/GO-2 and CS/GO-3 respectively.

Table 2: Representing the release exponent (n) along with the mechanism of drug release

Release Exponent (n) Mechanism of drug release
n<0.45 Quasi Fickian diffusion
0.450.89 non-fickian Super Case II transport

The release profile of the scaffolds proposes two type of drug releases models according to the system involved-fickian (Case-I) and non-fickian models (Case-II, Anomalous Case and Super Case II). The n value in the table 2 indicates the mechanism of the drug release in which n value less than 0.45 indicates the fickian release, n is equal to 0.89 indicates Zero order release Case I transport mechanism whereas the n lies in between the values 0.45