Quality by Design (Qbd) Based Development and Evaluation of Carvedilol Loaded Polymeric Nanoparticles for Enhanced Solubility

By preparing a polymeric nanoparticle by nanoprecipitation using specific polymers like Chitosan and HPMC K15M and Poloxamer 407 as a surfactant, the drug solubility of Carvedilol, a BCS class-II drug with poor water solubility, can be improved through the release of drug over time. Critical quality parameters, such as drug release (%), entrapment efficiency (%), particle size (nm), and zeta potential (mV), are used to eliminate unnecessary process and formulation variables. The model drug has a sharp melting point


Introduction
Like ultrafine particles, nanoparticles (NPs) are 1-100 nanometers.Nanoparticles may or may not have size-related features different from bulk materials and tiny particles [1] .Polymeric nanoparticles are 1-1000 nm colloids.Pharmaceutically active compounds are embedded in macromolecules [2] .Due to the quantum size effect, polymeric NPs affect oxidative stress, cytotoxicity, and genotoxicity [3] .Nanoprecipitation requires two miscible solvents.An acetone or acetonitrile-dissolved polymer is in the internal phase.They evaporate easily because they are water-impermeable.This approach uses polymer interfacial deposition after organic solvent displacement from a lipophilic solution to the aqueous phase.An intermediate-polarity water-miscible solvent dissolves the polymer.The solution is added to an aqueous solution dropwise or at a regulated rate [4] .Due to the fast spontaneous diffusion of the polymer solution into the aqueous phase, nanoparticles form instantly to escape water molecules.As solvent diffuses from nano-droplets, polymer forms nanocapsules or nanospheres [5] .The organic phase is usually introduced to the aqueous phase; however, it can be reversed without affecting nanoparticle synthesis.Surfactants stabilize colloidal suspensions; however, they are not necessary for nanoparticle synthesis.Superior to emulsification solvent evaporation, the nanoparticles have a welldefined size and constrained size distribution [6,7] .
Absorbable polymeric nanoparticles that respond to internal or external stimuli improve therapeutic delivery to sick areas.Micelles, vesicles, cross-linked nanoparticles, and hybrid nanoparticles are used to make stimulus-sensitive polymeric nanoparticles [8] .Polymeric nanoparticles' chemical or physical characteristics change with single, dual, or multiple stimuli.This lets them hold payloads during circulation, target sick areas, and release them after cell internalization [9] .nline at: . The sympathetic nervous system signals via adrenergic receptors and propan-2-ol to promote breast cancer.Since this discovery, cardiac-blocker drugs are considered cutting-edge anticancer treatments.Due to first-pass metabolism and limited water solubility, this medication has 25% to 35% absolute bioavailability [10] .
This study developed and optimized nanoprecipitation-prepared carvedilol-loaded polymeric nanoparticles to improve medication solubility in a sustained release mechanism and reduce drug dose intervals.There are numerous ways to make nanoparticles, but nanoprecipitation is the most common since it involves the fewest steps.The following objectives were chosen to conduct pre-formulation studies: analytical method development to develop polymeric nanoparticulate systems using selected polymers to optimize formulation variables or conditions and characterize prepared formulations.

Materials And Methods
Reddy's Laboratories, Hyderabad, India, provided gift samples of pure drug sample (Carvedilol) and other gift polymer samples such as chitosan, HPMC K 15M, Poloxamer 407 for the academic research purpose.Sigma Aldrich, India, provided acetone.Throughout the study, all essential ingredients used in the research have been of excellent quality.

Solubility Analysis
10 mg of the drug was dissolved in 9 ml of 0.1 N HCl, 1 ml of ethanol was added as a co-solvent, and the volume was increased to 10 ml.The quantitative solubility studies of the drug (Carvedilol) were carried out using different solvents, i.e., water, acetonitrile, phosphate buffer 6.8 and 7.4, 0.1 N HCl, methanol, ethanol, DMSO, PEG 200 and 400, n-octanol.First, 5 ml of each solvent was taken, and a minute amount of the drug was added to its saturation point.Then it was placed in a shaker for about 3 h.After that, the solubility of the drug was noticed for all the solvents.If it is entirely soluble, then again, 2 ml of each solvent was taken, and the drug was added up to its saturation point, which was placed in the shaker for 3 h.Then filtration was carried out for the respective solvents and analyzed under a UV spectrophotometer.The solubility analysis data of pure drugs in different solvents are shown in Table-1 and Figure-1 [11] .

Critical quality characteristics (CQAs) and the quality target product profile (QTPP)
In a broader sense, QTPP refers to a drug's predetermined anticipated characteristics, which are necessary to establish the product's intended performance concerning safety, and efficacy further to enable the recognition of product CQAs.The QTPP was determined based on regulatory and scientific requirements as listed in Table-2.QTPPs, which regulate the development of goods and processes, create CQAs.They are also coupled to in-process materials like critical material attributes (CMAs) and process parameters like Critical Process Parameters (CPPs) in synthesizing nanomaterials [12] .

Optimization by response surface methodology
It was optimized using Design Expert 12.1.1.(State-Ease Inc., Minneapolis, MN).Two independent factors were considered: drug: polymer ratio (A), Stabilizer concentration (B), and the additional impact of these individual variables on observed responses such as drug release (%), entrapment efficiency (%), particle size (nm), and zeta potential (mV).Table-2 depicts the optimization design with the two components and three levels.The model described 13 different runs undertaken, and the responses for each run were documented.Finally, the composition with the optimal outcomes was chosen for future research.

Method of preparation of polymeric nanoparticles
Nanoparticles were made via solvent evaporation and nanoprecipitation.
Step one: drug ratios 1:1, 1:2, 1:3.20 mg drug: 20mg polymer, 40 mg drug: 80 mg polymer, 60 mg drug:180 mg of chitosan and HPMC K15M were dissolved in 5ml glacial acetic acid and acetone.The polymeric and drug solutions were combined using a magnetic stirrer after the pure drug carvedilol was dissolved separately in 10ml of acetone.From this, 20ml of the drug-polymer solution and 5ml of the solution were added to distilled water containing 1%, 1.5%, and 2% poloxamer 407 to make 20 ml.We used a rotary flask evaporator to evaporate the acetone under reduced pressure to 10ml.Slowly add the medicine to the polymer solution to dissolve it.The medication and polymer were gently added using a #27-gauge needle-size nline at: le o b ila Ava -2321 -syringe at 1 ml/min.The adding procedure should take 1hr at 1000 rpm.To remove acetone, the 20ml final volume was evaporated in a rotating flask.We adjusted the volume to 10ml.The nano suspension was freeze-dried for 36 h to produce polymeric nanoparticle powder.

Characterization Fourier transform infrared spectroscopy (FT-IR)
Triturate the solid (drug) with dry, finely powdered potassium bromide.The amount taken should be such that the weight of the substance per area of the disc.Insert a portion of the mixture in a special die and subject it under a vacuum to high pressure.Mount the resultant disc in a suitable holder.IR scans potassium bromide 45 times in 1.5 minutes.A blank spectra of air backdrop was collected before capturing the sample spectrum.A pure drug sample, a pure polymer sample, and formulations, including the drug and the polymer, were scanned (Figures-2 to 4) [13] .DSC studies assessed the interaction between the drug and the polymer.A thermogram of carvedilol, polymers, and poloxamer 407 was determined.The DSC curve of carvedilol showed an endothermic peak at 120.9 0 C, an onset temperature of 113.2 0 C, and an end set temperature of 125.5 0 C, corresponding to its melting point (Figures-5 to 7) [14] .

Entrapment Efficiency
Exactly 2 ml of the sample was taken from the respective formulations in a centrifuge tube.First, the centrifuge tube was taken, and centrifugation was performed at 8000 rpm for about 25 minutes.After 25 minutes, the centrifuge tube was carefully removed and observed for the formation of a supernatant layer above the sample.Next, the supernatant layer of the liquid, i.e., about 1 ml, was carefully transferred into a test tube, and the volume was made up to 10 ml (Ethanol and 0.1 N HCl).Then the sample solution was analyzed under UV at ʎmax 282 nm [15] .

Statistical analysis
Design-Expert® (Version 12), Stat-Ease Inc., Minneapolis, MN, advanced statistical software of USA, was employed for formulation optimization and the estimation of its critical method parameters (CMAs).Microsoft performed the data evaluations excels 2007 (Microsoft, USA).

Optimization of process variables
To explore the influence of formulation and preparative variables of the nanoprecipitation technique on the formation of nanoparticles and their size, the polymer type and concentration, selection of organic solvent, stabilizers and their concentration, and the ratio of solvent (S) to non-solvent (N.S.), etc. were studied to control and optimize the process.The drug and polymer ratio were varied to see the effect on drug release, entrapment efficiency, particle size, and zeta potential.To know the impact of stabilizer concentration and the temperature used during the formulation of nanoparticles and similarly, the ratio/proportion of the solvent and non-solvent was varied to see the effect on nanoprecipitation of carvedilol nanoparticles and the stirring speed on the formation of nanoparticles: While following the nanoprecipitation method the stirring speed was various to observe the impact on the appearance of nanoparticles [16] .

Particle size distribution and zeta potential determination
The droplet size and size distribution analysis were performed on optimized formulation by using Malvern Zeta sizer (Nano ZS-90 U.K).The statistical distribution of droplet size of optimized formulation is shown in the Figure -8.

In-vitro diffusion study
Initially, 150 ml of 0.1 N HCl was taken in respective beakers.Then, about 5ml of the sample (F1-F9) was taken from the individual formulations, loaded with the dialysis membrane bag, and carefully tied with the help of thread.Dip the membrane bag into the beakers containing 0.1 N HCl for the first 2h and then change the dissolution medium with phosphate buffer solution with pH 6.8.Adjust the dialysis membrane properly inside the solution and on the magnetic stirrer at the stirring speed of 100 rpm.At 0 h, pipette 2ml of the sample and transfer it to the centrifuge tube, then add 2ml of 0.1 N HCl into the beaker to maintain the sink condition.A similar process was repeated at 2h, 4h, 6h, 8h, 12 h, 18h, and 24 h.This process was repeated for at least 24 h, and the 2ml of the samples collected was further divided, where 1ml of the sample was taken out and added with 1ml of ethyl acetate.The above solution was vortexed for about 15 minutes in a cyclomixture and kept aside for about 15 minutes.The formation of the supernatant clear liquid layer was carefully removed into a test tube and subjected to drying in a water bath.After completely drying this test tube, it was further analyzed under UV by adding the respective solvent into the tube (Table -3

Results and Discussion
The UV spectroscopy determined the standard curve for the pure drug and the initial solubility analysis shown in Table -1.The linearity range was determined up to 80 µg/ml in 0.1 N HCl.Hence, it obeyed Beer Lambert's law in this concentration range.Pure drug characterizations for compatibility and melting point were carried out with different polymers and excipients with the help of FT-IR and DSC.
The DSC studies revealed no interaction between the medication and the polymer.Carvedilol-loaded polymeric nanoparticles were created successfully by the nanoprecipitation method.Initially, the technique was performed at 500 rpm stirring speed and 70 0 C temperature, where the formulations of batches 1, 2, and 3 showed a hazy appearance.Hence the rate was decreased to 1200 rpm and 37 0 C temperature; by doing so, the clear formulations of carvedilol nanoparticles were obtained at different concentrations.

Particle size analysis
The size and dispersion of nanoparticles play a crucial role in their adhesion and interaction with cells.The particle size was minimum at 263.8, i.e. (-1, 0) in Run 6, while the maximum particle size in 1329.35nm, i.e., at (

Zeta potential
Zeta potential is a scientific notion for electro kinetic potential in colloidal systems and is one of the essential properties playing a significant role in nanomedicine.Zeta potential can affect the physical and pharmacokinetic aspects of nanosystems in the body or may affect nanoparticle phagocytosis in the bloodstream.The zeta potential was maximum at -5.85mV at Run 11, i.e., (1,1), while the zeta potential was minimum at 24.2mV at Run 6.

Entrapment efficiency
In this work, the influence of various process parameters The EE is the proportion of the drug-loaded into polymeric matrices.The percentage of the entrapped drug was minimum at 35.448, i.e. (1,1) in run 11.The percentage of entrapped drugs was maximum in Run 6, i.e., 86.225%-i.e.(-1, 0).The zeta potential carried out for the respective formulations is significantly less, which may affect the stability of the formulation.

Differential scanning calorimetry
The differential scanning calorimetry was performed to determine the peak temperature, onset and end set temperature, and heat energy for the pure drug carvedilol, polymers stabilizer poloxamer 407, as shown in Figure -3.DSC study revealed no interaction between the drug and polymers used.

Optimization of process variables
It optimised preparation variables and conditions; Drug: Table-2 demonstrates how nanoprecipitation created carvedilol polymeric nanoparticles with polymer ratio (mg) and stabilizer concentration (gm %).Vortex mixing the polymer in PEG 400, a benign solvent, created the diffusing phase.The drug was carefully weighed, added to the polymeric solution, vortexed, and left to stand to generate an airfree, transparent solution.Non-solvent polymer and medicament were insoluble in stabilizer-containing aqueous dispersion phase.Adding 1 ml of the diffusing phase to 19 ml of the dispersion phase (nonsolvent) with a syringe positioned to insert the needle directly into the aqueous medium under moderate (1200 rpm) magnetic stirring at 35 0 C formed nanoparticles.The polymer crystallized shortly after the solvent diffused into the dispersion medium, entrapping the medicine.Due to interfacial turbulences at the solvent:non-solvent interface and complex and cumulated phenomena including flow diffusion and surface tension variations, the Marangoni effect produced nanoparticles quickly

Effect of the factor on CQA (% of drug release)
The Figures-9 and 10 counter (2D) and response (3D) plots responses elucidate the impact of observed responses % drug release upon the stabilizer concentration and drug: polymer ratio.The stabilizer concentration gradually increased (Low level coded value -1, 0 was embedded in the model), and there is a significant variation in % drug release characteristic.However, when the level changes from low to high (0, to +1), i.e., results in a significant increase in drug: polymer concentration, there is a prevalence of dark green colour region replicating the considerable influence upon % drug release of polymeric nanoparticles.

Effect of the factor on CQA (% of drug entrapment efficiency)
Figures-10 and 11 counter (2D) and response (3D) plots depict that improvement in the drug: polymer concentration upsurges the level of size aggregation, which retards the release behaviour.This, in turn, enhances an optimum % entrapment efficiency specified in the prevalence of the red region.For example, in the case of polymeric nanoparticles of carvedilol, prepared with HPMC K15M and chitosan polymers, it was perceived that an abundant, desirable sustain release profile of 24 h was achieved with a much lesser proportion of HPMC K15M in comparison to other polymers.

Figure 1 .
Figure 1.Bar graph showing solubility of pure drug in different solvents

Figure- 9 .Figure- 9 .
Figure-9.2D graph showing effect of X1 and X2 on percentage of drug release

Figure- 12 .
Figure-12.2D graph showing effect of X1 and X2 on particle size

Figure- 14 .
Figure-14.2D graph showing effect of X1 and X2 on zeta potential

Table - 2
. Design matrix for the experimental runs as per the central composite design and their assigned codes to the formulation variables

Table - 3
. In-vitro drug release data of F1-F13 formulations Quality by Design (Qbd) Based Development and Evaluation of Carvedilol Loaded Polymeric Nanoparticles for Enhanced Solubility Available online at: https://jazindia.com-2326 -

Table - 1). Supplementary Table-1
. ANOVA for quadratic model data on percentage of drug release