Development of Clindamycin Loaded Oral Microsponges (Clindasponges) for Antimicrobial Enhancement: In Vitro Characterization and Simulated in Vivo Studies

Abstract

Clindamycin phosphate (CLP) is a broad-spectrum antibiotic that is used widely for different types of infections. It has a short half-life and hence it should be taken every six hours to ensure adequate antibiotic blood concentration. On the other hand, microsponges are extremely porous polymeric microspheres, offering the prolonged controlled release of the drug. The present study aims to develop and evaluate innovative CLP-loaded microsponges (named Clindasponges) to prolong and control the drug release and enhance its antimicrobial activity, consequently improving patient compliance. The clindasponges were fabricated successfully by quasi-emulsion solvent diffusion technique using Eudragit S100 (ES100) and ethyl cellulose (EC) as carriers at various drug-polymer ratios. Several variables were optimized for the preparation technique including the type of solvent, stirring time, and stirring speed. The clindasponges were then characterized in terms of particle size, production yield, encapsulation efficiency, scanning electron microscopy, Fourier Transform Infrared Spectroscopy analysis, in vitro drug release with kinetic modeling, and antimicrobial activity study. Moreover, in vivo, pharmacokinetics parameters of CLP from the candidate formula were simulated based on the convolution method and in vitro–in vivo correlation (IVIVC-Level A) was built up successfully. Uniform spherical microsponges with 82.3 µm mean particle size with a porous spongy structure were evident. ES2 batch exhibited the highest production yield and encapsulation efficiency (53.75 and 74.57%, respectively) and it was able to exhaust 94% of the drug at the end of 8 h of the dissolution test. The release profile data of ES2 was best fitted to Hopfenberg kinetic model. ES2 was significantly (p < 0.05) effective against Staphylococcus aureus and Escherichia coli compared to the control. Also, ES2 displayed a twofold increase in the simulated area under the curve (AUC) compared to the reference marketed product.

INTRODUCTION

Clindamycin phosphate (CLP) is a lincosamide antibiotic having a broad spectrum antimicrobial activity against staphylococci, streptococci most anaerobic bacteria, chlamydia trachomatis, and certain protozoa.¹⁾ This antibiotic induces its action by the inhibition of bacterial protein synthesis at the level of the 50S ribosome. High concentrations of CLP are achieved in most tissues including neutrophils, bones (60%), and joints (85%) but not in the central nervous system.²⁾ The oral bioavailability of CLP is non-linear and declines with increasing doses. The absolute bioavailability is 53 ± 14% after a 600 mg dose. In addition, its peak plasma concentration is 45 min, and a short elimination half-life of about 2 to 3 h.³⁾ Therefore, CLP should be administered every six hours to ensure adequate antibiotic blood concentration.

The oral dosage of CLP is 150–300 mg every six hours (q6h) for moderately severe infection and 300–450 mg q6h for severe infection. In addition, some of the more common side effects associated with oral administration of clindamycin include anorexia, esophagitis, rash, gastrointestinal tract (GIT) side effects, and metallic or unpleasant taste.⁴⁾ As a result, CLP is indicated when less toxic antimicrobial agents are not effective but with caution. Currently, CLP is only available in form of oral capsules, tablets, and topical solutions. Therefore, clindamycin has been one of the major interests for researchers though many years for the aim to improve the current marketed conventional formulations, they tried to incorporate it into multiple delivery systems like liposomes, nano-liposomes, nanofibers, nano-emulsion, and micro-emulsion to reduce its toxicity and antimicrobial activity.^5–7)

On the other hand, among the new delivery systems that are gaining broad attention are microsponges. These polymeric microparticles are extremely porous and can offer the prolonged controlled release of the drug moieties by entrapping them within their myriad of interconnected voids. Due to the small particle size of microsponges (particle size ranges from 5 to 300 µm), they can entrap high loads of drugs and exhibit unique compression and dissolution properties. Recently, microsponges have been designed to offer several advantages in pharmaceutical formulations, including delivering drugs to a specific target site of action,^8,9) altering drugs’ release patterns,¹⁰⁾ enhancing drugs’ stability¹¹⁾ reducing drugs’ mutagenicity and toxicity,¹²⁾ encapsulating natural products and herbs,¹³⁾ and improving the drug’s penetration through skin layers for topical administration.^14,15) Furthermore, other researchers have prepared topical gels containing clindamycin microsponges to treat acne¹⁶⁾ and other infectious diseases.¹⁷⁾ As of yet, no oral microsponges loaded with clindamycin have been reported.

Based on the previous introduction, the current research aims to prepare a new formulation of CLP-loaded microsponges (named Clindasponges) by using the quasi-emulsion solvent diffusion method for oral administration, as an attempt to improve clindamycin’s therapeutic activity, reduce its side effects and frequency of dosing which consequently enhancing patient’s compliance.

MATERIALS AND METHODS

Materials

CLP was purchased from Saga Laboratories Pvt. Ltd. (Ahmadabad, India). Eudragit S100 (ES100) was kindly gifted from Evonik Rohem GmbH (Germany). Polyvinyl alcohol (PVA), ethylcellulose (EC), dichloromethane (DCM), ethyl acetate (EA), disodium hydrogen phosphate, hydrochloric acid, and ethanol were obtained from Sigma-Aldrich (St. Louis, MO, U.S.A.). All the required materials for this work were used as procured and were of analytical grade.

Preparation of CLP-Loaded Microsponges

CLP-loaded microsponges were prepared by the quasi-emulsion solvent diffusion method¹⁸⁾ with some modifications. Two polymers, EC and ES100 were used to prepare the inner phase. The polymers were dissolved in two different solvents to prepare their solutions, where ES100 and EC were dissolved in DCM and EA, respectively. Then CLP powder was added to the polymer solution with gradual stirring on a magnetic stirrer at 1000 rpm and 25 °C. The internal phase was then poured into the PVA aqueous solution (0.5% (w/v)). After three hours of stirring, the clindasponges were formed in the form of white fluffy dispersed particles. The microsponges were then filtered, washed with Distilled water (D.W.), and dried in an oven (Sisco Instruments, India) at 40 °C for 24 h. Figure 1 describes the preparation technique of clindasponges and the compositions of clindasponges formulations are given in Table 1.

Fig. 1. A Scheme Describes the Preparation Technique of Clindasponges

Table 1. Compositions of Clindasponges Formulations

Formula code	Drug : polymer ratio (w/w)	ES100 (mg)	EC (mg)	DCM (mL)	EA (mL)	PVA* (g)
EC1	1 : 1	—	100	—	20	0.5
EC2	1 : 3	—	300	—	20	0.5
EC3	1 : 5	—	500	—	20	0.5
ES1	1 : 1	100	—	20	—	0.5
ES2	1 : 3	300	—	20	—	0.5
ES3	1 : 5	500	—	20	—	0.5

*The concentration of PVA solution is 0.625% (w/v). Each formula contained 100 mg of CLP.

Optimization of the Preparation Conditions of Clindasponges

To optimize the conditions of the clindasponges preparation technique, several factors have been studied including the type of solvent, time of stirring, and speed of stirring. This study was modeled by trial and error where the optimized conditions for microsponges preparation were decided based on the effect of the production yield percent and drug entrapment efficiency.

Characterization of Clindasponges

Production Yield

To determine the production yield of the clindasponges, the initial raw materials and the final product of each formula were weighed accurately, and the percentage yield was calculated using the following formula:

  

(1)

Percentage Entrapment Efficiency Analysis (EE%)

A specified weight (0.1 g) of each formulation was dispersed in a mixture of ethanol and phosphate buffer (1 : 4 v/v) and stirred for 30 min on a magnetic stirrer at 25 °C. The samples were centrifuged at 5000 rpm for 15 min to separate the unentrapped free drug from the clindasponges. The samples then were filtered using a Whatman^® filter paper (Sigma-Aldrich), diluted with phosphate buffered saline (PBS) (pH 7.4), and analyzed for the drug content using Shimadzu UV-1800 spectroscopy (Shimadzu, Kyoto, Japan) at λ_max 210 nm. The concentration of CLP was determined from the linear regression equation (y = 3.145x + 0.057; R² = 0.9992) of the previously constructed calibration curve of CLP in PBS (pH 7.4). Equation 2 was implemented to calculate the EE% of the prepared formulations, as given below:

  

(2)

Particle Size Measurement

The mean particle size and particle size distribution (PDI) of CLP-loaded and blank microsponges were measured at 25 °C using Malvern Zeta-Nanosizer (Worcestershire, U.K.). The particle size measurement was performed after diluting 1 mL of each sample in 10 mL of D.W. The samples were placed in the disposable plastic cuvette inside the instrument and stabilized at an appropriate angle of 173°. Each measurement was carried out in triplicate and the results were presented as mean ± standard deviation (S.D.).¹⁹⁾

In Vitro Drug Release

The in vitro release of CLP from the microsponges was investigated for all formulations using the Franz cell diffusion system. The system was designed as follows: a receptor chamber was filled with the dissolution media (22 mL of the simulated gastric fluid (SGF) pH 1.2, and phosphate buffer pH 6.8). The Millipore^® membrane (0.45 µm pore size) was placed on the top of the chamber. One milliliter of each formulation was placed on the membrane in form of an aqueous dispersion (1 mg/mL). The whole system was maintained at 37 ± 0.5 °C and 150 rpm. The run was carried out for 2 and 8 h in the acidic medium and phosphate buffer, respectively. At regular intervals, samples (3 mL) were withdrawn, and the depleted volume was compensated with an equal volume of a freshly prepared medium. Samples were analyzed for the drug content spectrophotometrically at a wavelength of 210 nm.

Drug Release Kinetics Analysis

Data obtained from the drug release profiles were analyzed using graphical presentation and model-dependent methods. The DDSolver Excel add-in software was used for the kinetics studies and data analysis. The model-dependent method was conducted to describe the dissolution profiles based on different mathematical equations (Table 2), including Zero-order, First-order, Higuchi, Hixon Crowell, Hopfenberg, and Korsmeyer-Peppas models.²⁰⁾ The coefficient of determination (R²) has been used to describe the release pattern of the optimized formulas. The R² of all the models was compared to find the best model that best fitted the drug release data. Then, the % of fraction dissolved for both the observed data and the predicted data were computed, compared, and presented as line charts by the use of DDSolver Excel add-in software.²¹⁾

Table 2. Model-Dependent Mathematical Equations

Kinetic model	Mathematical equation
Zero-order	Q = Q0 + K0t
	where Q is the amount of drug dissolved in time t, Q0 is the initial amount of drug in the solution and K0 is the zero-order release constant.
First-order	Log Ct = Log C0–kt/2.303
	where C0 is the initial concentration of the drug, k is the first-order rate constant, and t is the time.
Higuchi
	where Q is the amount of drug dissolved in time t, and KH is the Higuchi dissolution constant.
Hixon Crowell	M01/3-Mt1/3 = κt
	where M0 is the initial amount of drug in the pharmaceutical dosage form, Mt is the remaining amount of drug in the pharmaceutical dosage form at time t and κ (kappa) is a constant incorporating the surface-volume relation.
Hopfenberg	Q = KH*t1/2
	where Q is the amount of drug dissolved, t1/2 is the time it takes a drug to lose half its original concentration, and KH is the Hopfenberg dissolution constant.
Korsmeyer–Peppas	Mt/Mα = Ktn
	where Mt/Mα is a fraction of the drug released at time t, k is the release rate constant, and n is the release exponent.

In Vivo Data Prediction (Convolution Method)

The CLP in vitro release data from the optimized formulas (ES1 and ES2) was converted into a drug plasma concentration-time profile through the convolution method. The cumulative drug release (%) values gained from the in vitro dissolution test was converted into discrete amount (mg) within every sampling time. The amount of drug eliminated with time was calculated for every amount segment using the first-order elimination rate Eq. 3:

  

(3)

Then, the total amount of drug present in the blood at different times was calculated by adding all the calculated drug amounts for every time interval. Lastly, the plasma concentration of CLP (µg/mL) was calculated by multiplying the blood amount of each time interval by the drug bioavailability and then dividing it by the drug volume of distribution (Vd), as shown in the Supplementary 1 (a, b).²²⁾

CLP Pharmacokinetic Parameters

The pharmacokinetic (PK) parameters of CLP were obtained from well-authentic published papers.²³⁾ The bioavailability of CLP is 40–50%, it was used in the calculations as 0.45, the half-life is 3 h, and the volume of distribution is 1–1.3 L. Accordingly the elimination rate constant (Ke) was found as 0.231 h⁻¹. Further PK parameters were gained depending on the predicted in vivo data such as C_max, T_max, and AUC. %F also has been calculated by using the following formulas:

  

(4)

  

(5)

In Vitro–in Vivo Correlation (IVIVC) Study

Level A correlation was built up successfully by plotting in vitro drug release (%) versus the predicted (simulated) plasma concentration (µg/mL) of CLP. The time points of the plasma concentration were (0, 0.5, 1, 2, 3, 4, 5, 6, 8, 10, 12, …, 24 h). The linear regression coefficient (R²) was obtained and compared between ES1 and ES2.

Morphological Examination

The surface topography and morphological characteristics of the optimal clindasponges formula (ES2) and the blank microsponges were examined using a scanning electron microscope (SEM). On a clean slide cover, a specific weight (20 mg) of the sample was dispersed and allowed to dry under a vacuum. As a next step, the dried sample was fixed on a metal stub with conductive tape, and the target was coated with gold-palladium film using a Mini Sputter Coater (SC7620, Quorum Technologies, Laughton, U.K.). The SEM photographs at 3 kV were taken using the Thermo Scientific Apreo field emission SEM (Thermo Scientific, Waltham, MA, U.S.A.).

Fourier-Transform IR Spectroscopy (FT-IR) Spectroscopy Analysis

The purpose of the FTIR spectroscopy study was to assess any possible interactions between the drug and the polymer. Samples of the pure CLP, ES100 polymer, CLP-ES100 physical mixture, optimal microsponges formula (ES2), and blank of the optimal formula (drug-free microsponges) were scanned using a JASCO FTIR 6300 spectrophotometer (JASCO, Easton, U.S.A.). A hydraulic press was used to compress the samples into KBr pellets using 5 t of pressure for 5 min, and then spectra were recorded at a resolution of 4 cm⁻¹. The scanning range was from 4000 to 400 cm⁻¹.

Antimicrobial Activity Study

The antimicrobial activity was tested according to the CLSI guidelines.²⁴⁾

Preparation of the McFarland Standard

McFarland standard solution was prepared by adding 0.05 mL of 1% (w/v) solution of barium chloride and 9.95 mL of 1% (v/v) solution of H2SO4 and mixed with continuous stirring. The standard solution was stored in sterile screw cap tubes of the same sizes. The screw cap tubes were tightly closed to avoid any contamination and loss by evaporation. They were stored at room temperature and protected away from sunlight.²⁵⁾

Nutrient Broth

For inoculation and sub-culturing of bacteria, the nutrient broth was prepared according to the specified procedure provided by the manufacturer (23 g in 1 L D.W.). Four milliliters of each of the prepared Nutrient broth was transferred to numerous test tubes. The test tubes with the broth were then autoclaved at 121 °C for 15 min at 15 lb pressure.²⁵⁾

Nutrient Agar

For the sub-culturing of the organism Staphylococcus aureus and to perform the antibiotic susceptibility tests, Nutrient agar was prepared according to the procedure as stated by the company (28 g in 1 L D.W.). The agar flask was then autoclaved at 121 °C for 15 min at 15 lb pressure.²⁵⁾

Inoculum Preparation

Inoculum was prepared by using broth suspension of isolated colonies selected from a 24-h inoculated agar plate. The suspension was adjusted to attain turbidity corresponding to 0.5 McFarland turbidity standard. Within the time of 15 min of preparation, the adjusted inoculum suspension is diluted in broth so that the corresponding bacterial population is approximately 5 × 10⁵ CFU/mL.²⁵⁾

Antibiotic Susceptibility Testing/Diffusion Technique

The Antibiotic susceptibility testing was done according to the CLSI guidelines.²⁵⁾ Antibiotic susceptibility test using diffusion technique measures the zone of inhibition diameters which provides estimates of the susceptibility of bacteria to antimicrobials. This procedure uses a Well diffusion technique, where 1% of CLP and formulated 1% equivalent drug CLP incorporated in microsponges in suspension was used to test the susceptibility of Gram-positive Staphylococcus aureus (Sample No. ATCC^®-25923™) and Gram-negative bacteria Escherichia coli (ATCC^® 25922™). The test is performed by applying a bacterial inoculum of approximately 5 × 10⁵ CFU/mL to the surface of a large (150 mm diameter) Nutrient agar Petri^® plate by using the pour plate technique. The diameter of the well was 7 mm and 50 µL of the antibiotic solutions were added to the well then, the plates were incubated at 37 °C for 24 h. The zone sizes were measured in mm with a Kirby Bauer standard scale.²⁶⁾

Statistical Analysis

Statistical tests were conducted using IBM SPSS^® statistics version 1.0.0.1508. A mean value and an S.D. were used to express all results (n = 3). Student t-test and Oneway ANOVA test had been used for data comparison and the differences at a level of 0.05 were considered to be statistically significant.

RESULTS AND DISCUSSION

Optimization of the Preparation Process

In this work, two different solvents namely, DCM and EA, had been used in the preparation of Clindasponges to dissolve ES100 and EC polymers, respectively. In our preliminary trials for the optimization of the preparation process, the results (not shown) proved that the type and amount of the solvent in the formulations significantly affected the production quality and quantity of the Clindasponges. The amount of 20 mL of DCM (in ES100-clindasponges) and EA (in ES100-clindasponges) was the best to produce uniformly dispersed porous microparticles with optimized loading efficacy. These results were in agreement with those of Pawar et al., who found that the type and concentration of solvent were critical for the preparation and stabilization of oxybenzone-loaded microsponges in form of topical gel.²⁷⁾ In addition, microsponges prepared with DCM solvent exhibited higher entrapment efficiency than those prepared with EA. This may be due to the rate of solvent evaporation where EA evaporated so quickly before the appropriate creation of microsponges, resulting in a lower drug loading. Another study performed by Murthy and Kishore also pointed out that different solvents might influence drug diffusion from the microsponges, consequently reducing their loading into the carrier.²⁸⁾

Furthermore, the study found that two additional variables, the speed of stirring and the time of stirring, played a role in the yield and quantity of the fabricated microsponges. The yield production and drug entrapment efficiency of the prepared formulations increased with higher speeds of stirring (Fig. 2). This could be due to the uniform dispersion of drug particles in the polymer solution, preventing the formation of agglomerates thus more drug was entrapped during the process.²⁹⁾ On the other hand, there was no significant (p < 0.05) effect of stirring duration on EE%, however, 1.5 h of stirring was not sufficient for complete solvent evaporation, whereas 3 h of stirring was optimal.³⁰⁾

Fig. 2. The Entrapment Efficiency of Clindasponges at Various Stirring Speeds and Stirring Times

* Results are significant at p < 0.05.

Evaluation of Clindasponges Formulations

Particle Size Measurement

The average of the prepared Clindasponges was in a range between 66.6–110 µm (Table 3). The diameter of these spherical particles increased when the polymer’s amount in the formula increased. Probably, this elevation is a result of the condensed polymer surrounding the drug in each particle giving the larger appearance. Additionally, the percentage PDI value was less than 30% for the analyzed Clindasponges formulas indicating their size uniformity and homogeneity.

Table 3. Production Yield, %EE and Particle Size of Clindasponges Formulations

Formula code	Production yield (%)	EE (%)	Particle size (µm)	PDI (%)
EC1	40.27±1.5	42.96±1.1	66.6±0.91	11.2
EC2	54.54±8.6	59.89±4.9	69.8±1.19	9.8
EC3	64.98±4.2	69.28±8.7	107.5±7.14	2.4
ES1	44.70±1.9	56.78±6.7	68.3±5.21	7.8
ES2	53.75±2.7	74.57±2.2	71.6±4.83	2.5
ES3	77.12±6.3	93.04±1.5	110±4.32	8.6

Results are given as mean ± S.D. (n = 3).

Production Yield and EE

All batches of microsponges had production yield in the range of 40–77%, as shown in Table 3. Both EC1 and ES1 possessed the lowest production yield whereas EC3 and ES3 showed the highest values. The difference in percentage yield is due to the drug : polymer ratio. The greater the drug: polymer ratio 1 : 5 in EC3 and ES3, the greater the production yield, 64 and 77% respectively. This is due to the longer path provided by dense polymer layers for solvent evaporation to the aqueous phase hence extra time for unique droplet formation subsequently enhancing yield. However, reducing the drug: polymer ratio 1 : 1 in EC1 and ES1 resulted in 40 and 44% yield, respectively. Similarly, %EE has the same relation to drug: polymer ratio as any increase in polymer amount provides more time for fabrication of microsponges and better entrapment. The EC3 and ES3 formulations entrapped the drug more effectively than EC1 and ES1 which had lesser polymer amounts.

In Vitro Release Studies of Clindasponges

The in vitro release of CLP from the Clindasponges was studied in both acidic and basic dissolution media. The drug release in the SGF was not exceeding 18% in the first hour and remained at a plateau for the rest of the run. This may be attributed to the basic nature of CLP (pK_a 7.56) which limited its release from the anionic matrix of the microsponges into the acidic medium.

However, it was observed that the dissolution of CLP in the phosphate buffer was influenced by the type and ratio of polymers included in the formula (Fig. 3). The prepared CLP microsponges exhibited different patterns of drug release where EC1, EC2, and EC3 exhibited the least drug release compared with ES1, ES2, and ES3. It has been noted that ethyl cellulose batches (EC1, EC2, and EC3) had a very poor drug release over time therefore no further investigations were carried out for the above-mentioned formulations because of the inadequacy and release of CLP from the matrix of EC-based Clindasponges.

Fig. 3. In Vitro Release Profiles of CLP from Clindasponges Formulations in PBS pH 7.4

The error bars represent the minus-plus S.D. from the mean (n = 3).

Moreover, relying on Fig. 3, it was remarkable that the release of CLP from ES1, ES2, and ES3 had been reduced from 96 to 80% with the increase in drug: polymer ratio from 1 : 1 to 1 : 5. Such a decrease can be justified by the diffusion path facts. The high amount of polymer retarded the drug release by increasing the thickness of the matrix formed and thus prolonging the diffusion path of the drug and decreasing the release. The profiles showed that the ES1 formulation gave a significantly higher release of the drug over time while ES3 had the lowest and the most sustained. Additionally, the overall release of the drug from the microsponges was well-controlled throughout the dissolution run, eight hours.

The release pattern of the drug from the prepared matrixes was investigated also by using an Excel sheet add-in tool. Various release kinetics Zero order, First order, Higuchi, Korsmeyer–Peppas, Hopfenberg, and Hixon Crowell were analyzed for ES1, ES2, and ES3 to conclude the best fitting model depending on the R² value. The release pattern analysis (Table 4) revealed that all formulations were following the zero-order, Higuchi, Hopfenberg, and Korsmeyer-Peppas models. The R² for these models was more precise for ES1 and ES2 formulations in comparison to ES3.

Table 4. Release Kinetics Data of Clindasponges Formulations

Batch code	Zero-order		First-order		Higuchi		Korsmeyer–Peppas			Hixson Crowell		Hopfenberg
	k0	R2	k1	R2	KH	R2	n	k	R2	khc	R2	kHB	R2
ES1	12.08	0.98	0.21	0.90	28.6	0.98	0.97	13.3	0.98	0.06	0.95	0.14	0.99
ES2	12.5	0.98	0.20	0.92	27.9	0.97	0.99	12.5	0.97	0.06	0.94	0.15	0.99
ES3	9.5	0.98	0.12	0.93	20.6	0.92	1.29	5.6	0.98	0.04	0.90	0.11	0.97

The Zero-order release model indicated a sustained release of the drug from the microsponges matrixes.¹³⁾ While the Higuchi model represented the diffusion mechanism that can be a result of the proper formation of the heterogenous and spherical microsponges matrixes.³¹⁾ Moreover, the Hopfenberg model suggests that the drug release from the microsponges can be because of microsponges’ surface erosion.³²⁾

While in the Korsmeyer–Peppas model, the n value represents the mechanism of the drug release from matrix systems. The “n” value equal to 0.5 refers to a Fickian diffusion-controlled release mechanism, while the n value equals to 1.0 refers to a non-Fickian diffusion-controlled release (case II transport), and the system approaches Zero-order drug release. On the other hand, if the n value is between 0.5 and 1, it is assumed that the release mechanism is governed by both Fickian diffusion and case II transport as in ES1, ES2, and ES3. The results of the current research results in agreement with those previously reported by Shahzad et al. who studied the effect of a combination of hydrophobic and hydrophilic polymer ratios on controlled drug release from cellulosic microsponges.³³⁾

Furthermore, the % of fraction dissolved versus time of the observed data was compared with the predicted data and the results explored a very good association as in Fig. 4. Thus, the sustained release behavior of the prepared microsponges was confirmed and the release of the drug was a result of both diffusion and surface erosion mechanisms.

Fig. 4. Percentage of Fraction Dissolved vs. Time According to First-Order Kinetics for ES1, ES2, and ES3

In-vivo Data Prediction (Convolution Method)

The convolution-based IVIVC methods offer several advantages such as the direct prediction of the plasma concentration-time course and the ability to predict measured quantities, not indirectly calculated quantities such as the cumulative amount absorbed. The utility of product-specific IVIVC for oral products has been well documented in the literature.²²⁾ Clindamycin belongs to class III based on the “Biopharmaceutical System Classifications” (BCS) due to its high solubility, and low permeability with a protein binding of 95%.³⁴⁾ Therefore, research is needed to improve its absorption, and thus its bioavailability.

In the present work, the simulated CLP plasma concentration–time profiles for ES1 and ES2 (Fig. 5) that were constructed from the in vitro data exhibited a lower bioavailability profile for ES1. The pharmacokinetics parameters of ES1 and ES2 were compared with a reference³⁵⁾ and C_max for ES1 and ES2 was found to be 1.93 and 2.71 ng.mL⁻¹ respectively, while the reference stated the C_max as 3.02 ng.mL⁻¹. The AUC of all the formulations was significantly higher (p < 0.05) than the reference AUC. ES2 showed a twofold increase in the AUC and thus the bioavailability, as F% is 189% for ES2 (Table 5). In the current study, the Clindasponges strength was equivalent to 100 mg of the pure drug. If the strength of CLP is increased to 300 mg, as in the commercial product, the C_max will surpass the minimum inhibitory concentration (MIC) of CLP.³⁶⁾

Fig. 5. Simulated CLP Plasma Concentration–Time Profiles of ES1 and ES2 Clindasponges

The error bars represent the minus-plus S.D. from the mean (n = 3). * Results are significant at p < 0.05.

Table 5. DDSolver-Simulated Pharmacokinetic Parameters of ES1 and ES2 Clindasponges Formulations

PK parameters	ES1	ES2	Reference product
Cmax (ng.mL−1)	1.934	2.71*	3.02
AUC0–24 (ng.h.mL−1)	14.94	20.44	10.3
AUC0–∞ (ng.h.mL−1)	15.77	21.18*	11.2
%F0–24	145.04	198.44*	—
%F0–∞	140.8	189.1*	—

* Results are significant at p < 0.05.

In addition, the IVIVC level-A correlation for ES1 and ES2 was built up using the in vitro drug release (%) versus the predicted (simulated) plasma concentration (µg/mL) of CLP. The R² for the constructed linear charts was 0.976 and 0.989 for ES1 and ES2 thus considered an excellent relationship,³⁷⁾ as displayed in Fig. 6. The level A correlation of the IVIVC has been achieved successfully in the development of divalproex sodium III as well.³⁸⁾

Fig. 6. IVIVC Level-A Correlation for ES1 and ES2 Clindasponges

In conclusion, ES2 clindasponges exhibited an acceptable sustained release pattern in the in vitro drug release study, with a high cumulative release percentage, and explored the highest bioavailability hence selected as the optimal formula and subjected to further investigations including FTIR spectroscopy, SEM test, and in vitro antimicrobial studies.

Scanning Electron Microscopy

The SEM test was conducted to characterize the morphology and surface topography of the vesicular particulates, as well as to confirm drug loading into these entities.³⁹⁾ The captured SEM images at different powers of magnification revealed that the optimal formulation ES2 clindasponges are predominantly spherical with a fine uniform structure (Figs. 7A, B). The high porosity of the surface of these microparticles was also observed and the average pore size of the particles was measured and found to be 584.63 µm (Fig. 7C). The findings also suggested a significant (p < 0.05) correlation between the drug: polymer ratio and clindasponges porosity, where the increase of this ratio decreased the porosity of the particles and consequently the release rate of CLP from the microsponges.

Fig. 7. SEM Images of ES2 Clindasponges at A) 300X, B) 1600X, and C) 4000X Power of Magnification

FT-IR Spectroscopy Analysis

This instrumental analysis was employed to investigate drug-polymer compatibility.⁴⁰⁾ The characteristic FT-IR peaks of pure clindamycin phosphate were C–CL stretch at 640 cm⁻¹, C–N stretch at 1042 cm⁻¹, C–O stretches at 1150 cm⁻¹, C–O–H bonding at 1450 cm⁻¹, C=O stretch at 1682 cm⁻¹, C–H stretch at 2921 cm⁻¹ and N–H stretches at 3264 cm⁻¹. These principal peaks were observed in the physical mixture (CLP and ES100) and the prepared microsponges (Fig. 8). Thus, the FT-IR study of CLP, physical mixture, and clindasponges did not show any major shift of peaks, indicating no chemical interaction between the drug and polymer. These results depicted complete compatibility and expected constant good stability in the optimized microsponges.

Fig. 8. FT-IR Spectra of A) CLP, B) Physical Mixture, C) ES2 Microsponges, D) CLP-ES2 Physical Mixture

In Vitro Antimicrobial Study

Antibiotic susceptibility test was performed on 1% marketed clindamycin drug and 1% w/v equivalent formulated clindamycin drug incorporated in microsponges in suspension form, using the standard diffusion method following the CLSI guidelines. Fifty microliters of formulated drug and marketed drug solution were applied to evaluate their susceptibility testing against two microorganism Staphylococcus aureus and Escherichia coli respectively.⁴¹⁾ The experiments were repeated three times. The results were expressed as a zone of inhibition in mm ± S.D. and are shown in Table 6.

Table 6. Zone of Inhibition (mm) ± S.D. of Prepared Clindasponges (ES2) Compared with the Blank ES2, Reference and Control Suspensions

Drug	Zone of inhibition (mm) against Staphylococcus aureus (mean ± S.D.)			Zone of inhibition (mm) against Escherichia coli (mean ± S.D.)
	16 h	18 h	24 h	16 h	18 h	24 h
Reference CLP suspension 1% (w/v)	36.5 ± 0.71	36.5 ± 0.71	37.5 ± 2.12	11 ± 1.41	13 ± 1.41	13 ± 1.41
Positive Control	37.5 ± 0.71	38.5 ± 2.12	39.5 ± 0.71	10.5 ± 0.71	12 ± 0.0	12 ± 0.0
Blank ES2 (Clindasponges)	0.0	0.0	0.0	0.0	0.0	0.0
(ES2 Clindasponges) equivalent to 1% (w/v) CLP) suspension	47 ± 0.0	48 ± 2.83	49 ± 1.41	29 ± 1.41	34.5 ± 0.71	35 ± 0.0

A clear zone of inhibition against both Gram-positive and Gram-negative organisms was observed. By comparing the zone width of the Staphylococcus aureus strain zone with that of Escherichia coli strain zones it was observed that Staphylococcus aureus has a more zone width for both marketed and microsponges incorporated 1% Clindamycin in the suspension form than that of Escherichia coli strain zones for both marketed and microsponges incorporated 1% equivalent Clindamycin. Moreover, the marketed 1% (w/v) clindamycin has shown the lowest inhibitory activity against Staphylococcus aureus (36.5 mm ± S.D.) while on the other hand, the formulated 1% (w/v) clindamycin incorporated microsponges have shown a higher inhibitory activity (49 ± 1.41 mm ± S.D.) against Staphylococcus aureus.

In addition, previous studies had indeed implemented the same strategy of loading various antimicrobial agents into the nano/microcomposite particulate systems to improve their therapeutic efficacy. For example, an inhalable arbekacin-loaded nanocomposite coated with hydrophobic surfactants was prepared using a spray dryer for the treatment of Pseudomonas aeruginosa infectious disease.⁴²⁾ Also, Takishima and his group fabricated chitosan-coated EC microparticles for prolonged intestinal absorption of cephradine.⁴³⁾

CONCLUSION

The concept behind developing a polymeric microsponges delivery system was to deliver Clindamycin persistently for a long period to reduce application frequency, and hypersensitive reactions and to improve bioavailability, and safety than the marketed conventional formulation. The method implemented was quasi-emulsion solvent diffusion; found to be simple, reproducible, and rapid. The method resulted in microsponges of uniform spherical particle size and a porous spongy matrix. Drug polymer compatibility study showed no major interaction. Further, it has been noted that drug to polymer ratio had a consequential effect on particle size, production yield, and encapsulation efficiency. Among all prepared clindasponges by using different polymers in different ratios, Eudragit S100 proved its applicability when used in a ratio of 1 : 3 (drug: polymer), and a sustained release of 80% over 8 h was achieved. The in vitro drug release reflected the highest regression value for zero order, Higuchi, and the Hopfenberg models kinetics. The bioavailability has increased twofold and the prepared microsponges were effective against two microorganism Staphylococcus aureus and Escherichia coli. Thus, the developed microsponges are promising oral drug delivery systems providing a better mode of treatment over the present conventional antibiotic. This study provides future insights into the fabrication of different antibiotics using microsponges and raising the generally accepted standards in the antibiotic industry by solving the enormous problems associated with their consumption.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

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