Transdermal patches based on chitosan/hydroxypropyl methylcellulose and polyvinylpyrrolidone/hydroxypropyl methylcellulose polymer blends for gentamycin administration

ABSTRACT

Biofilm-forming bacteria have sent alarms to the world about the emerging of bacterial resistance. Gentamycin is an aminoglycoside broad-spectrum antibiotic used against microbial infections. The transdermal drug delivery method is a major system used to reduce drug toxicity and avoid first-pass metabolism. Gentamycin was formulated in a transdermal polymeric formula using hydroxypropyl methylcellulose (HPMC), polyvinylpyrrolidone (PVP), and Chitosan in the presence of palmitic acid as a permeation enhancer. In this research, gentamycin extended drug release behavior was successfully done in different polymeric formulas containing (HPMC/PVP) and (HPMC/Chitosan), with a maximum drug release of <70%. In addition, drug diffusion was found to be dependent on the rate of drug release. The controlled release formulas selected for antimicrobial assay show that HPMC/Chitosan formulas have successfully inhibited microbial and biofilm growth by 90%. In conclusion, gentamycin can be formulated in a transdermal polymeric film to target skin infection, reduce drug side effects and avoid drug first-pass metabolism.

INTRODUCTION

Over the past years, pharmaceutical companies and research facilities have been paid to develop a dosage form to overcome the serious threat to human life caused by bacterial infections.[1] Bacteria with the ability to form a biofilm layer, have antibiotic resistance a hundred times more than regular floating bacteria.[2,3] Researchers tried to formulate antibiotics in a polymeric matrix, with suitable release and diffusion profiles, but most of them have resulted in a burst drug release.[4] Transdermal drug delivery systems are dosage forms designed by embedding an active drug inside a polymeric matrix to control its release, prevent drug first-pass metabolism, and avoid drug fluctuation in the systemic circulation.[5] The ability of the transdermal patches to control drug release and to ensure drug penetration is affected by two factors, the polymeric matrix components, and the permeation enhancers.[6] Gentamycin is an aminoglycoside antibiotic used against skin infections caused by different biofilm-forming Gram-positive and Gram-negative bacteria.[7] The severe side effects associated with the long use of gentamycin such as ototoxicity, nephrotoxicity, and neurotoxicity have pushed the pharmaceutical industry to formulate it in a transdermal patch.[8,9] Hydroxypropyl methylcellulose (HPMC) is a cellulose polymer used in different controlled-release formulas, with the ability to carry hydrophilic drugs.[10] Chitosan has magnificent functional and biological properties that suit pharmaceutical and biomedicine research and formulas.[11] Polyvinylpyrrolidone (PVP) is an amorphous polymer used widely in cosmetics due to its safety and compatibility with other polymers.[12] The objective of this research is to formulate gentamycin in a biodegradable transdermal patch to see its ability to inhibit bacterial growth with an acceptable drug release and diffusion profile through different analysis techniques.

MATERIALS AND METHODS

Materials

99.9% pure gentamycin powder 99.9% pure, from MS Pharma for the pharmaceutical industry, Amman, Jordan. Acetonitrile-high-performance liquid chromatography (HPLC) grade was obtained from Sigma-Aldrich Company, Bengaluru, India. Palmitic acid, Chitosan, HPMC-K100M, and PVP were purchased from Sigma-Aldrich, Poznan, Poland. Biofilm forming bacteria Staphylococcus aureus (S. aureus) American Type Culture Collections (ATCC) 9144 and Pseudomonas aeruginosa (P. aeruginosa) ATCC 15442 were obtained from ATCC. Methanol, water, and phosphate buffer were obtained from Sigma-Aldrich, St. Louis, USA.

Methods

Polymeric film formation and drug loading

The polymeric films were prepared by dissolving the selected ratio of each polymer with 60 mL methanol and water 1:1. Mixed using a magnetic stirrer at 25°C followed by the addition of 100 mg of gentamycin wisely, and 2 mL of palmitic acid as a permeation enhancer.[13] The mixing was done for 5 h to have a homogeneous mixture and left under the fume hood for 2 days to complete drying. All polymeric films ratio with their codes is presented in Table 1.

Table 1.

Code and composition of gentamycin and polymers in each patch

Formula code	Drug weight (mg)	HPMC (%)	PVP (%)	Chitosan (%)
HPMC/PVP mixture
A1	100	100	0	-
A2	100	80	20	-
A3	100	20	80	-
A4	100	50	50	-
HPMC/chitosan mixture
B1	100	80	-	20
B2	100	20	-	80
B3	100	50	-	50

HPMC: Hydroxypropyl methylcellulose, PVP: Polyvinylpyrrolidone

Polymeric film characterization

Film thickness

Each polymeric film thickness was measured using a micrometer from four different positions and the mean of each was calculated with its standard deviation.

Content uniformity

A triplicate of 1 cm² from each polymeric film-containing drug was dissolved in 1 mL of methanol, and HPLC was used to calculate the concentration.

Moisture content

A triplicate of each film was weighed and placed individually in a desiccator and kept for 24 h in the presence of sodium chloride. Then a final weight was taken, and moisture content was calculated using the equation below. All mean and standard deviation percentages have been calculated.

High-performance liquid chromatography method

Content uniformity, drug diffusion, and drug release have been analyzed by Shimadzu Prominence-i LC 2030C HPLC instrument with an isocratic method. The mobile phases were composed of phosphate buffer (pH 7.4) and acetonitrile with a ratio of 30:70 respectively. The column used was ACS C18 (3 mm, 15 cm) with a flow rate of 1 mL/min at 27°C. All absorbance measurements were done under 245 and 290 nm wavelengths for both samples and calibration curves. Calibration curves resulted in a correlation coefficient of > 0.99.

Drug release and diffusion

For the release study, 1 cm² of each formula, in triplicate, was placed in 25 mL beaker containing 10 mL of phosphate buffer (pH 7.4) in 37°C room. Samples from each formula were taken at 5, 15, 30, 45, 60, 90, 120, 150, 180, 240, 300, 900, and 1440 min. At each time, 1 mL of phosphate buffer was added to maintain the volume. While for diffusion, diffusion apparatus with Franz cells was used containing 15 mL phosphate buffer (pH 7.4) at 37°C. Each sample was filtered and analyzed using HPLC techniques.

Antimicrobial assay

AlamarBlue® and planktonic assays have been used for the monitoring of microbial activity of the formulated gentamycin. A 1 cm² of each formula was placed in 6 well plates containing 1.5 mL of 1 × 10⁸ CFU of the tested bacteria. All plates containing samples were incubated for 16 h’s at 37°C followed by the addition of AlamarBlue® solution that will be oxidized in the presence of living cells. The plates were incubated for 2 h’s and absorbance reading was measured 560 nm excitation and 590 nm emission wavelengths. All percentages were calculated when compared to the positive control gentamycin at a concentration of 1 mg/mL. While planktonic assay solution was conducted using the similar step but without the addition of AlamarBlue® solution. After the incubation for 16 h’s patches were removed from the wells and each well was washed twice with 1.5 mL of phosphate buffer saline. Absorbance measurements were taken at 600 nm and percentages of biofilm formed were calculated when compared to the positive control 1 mg/mL gentamycin.

RESULTS

Polymeric films characterization

A triplicate of each formula was prepared, and an individual weight of each polymeric film was taken and presented in Table 2. The weights of the patches varied between 1094.3 and 1107.1 mg, with a standard deviation between 1.4 and 7.3 mg. In the same way, polymeric films’ triplicates show high similarity in the film thickness which varied between 11.4 and 12.1 μm, with a standard deviation of 0.17–1.05 μm.

Table 2.

Weight and thickness of gentamycin polymeric film, mean±standard deviation (n=3)

Formula code	Patch weight (mg)±SD	Patch thickness (µm)±SD	Content/cm2 (mg/cm2)±SD	Percentage moisture content±SD
A1	1095.3±1.7	11.7±0.32	1.21±0.42	0.71±0.92
A2	1107.1±3.2	11.6±1.05	1.23±0.27	0.83±0.75
A3	1095.7±2.7	11.7±0.43	1.19±0.49	0.69±1.01
A4	1101.8±1.4	11.4±0.92	1.21±0.31	0.53±0.37
B1	1091.2±7.3	12.1±0.17	1.27±0.37	0.42±0.31
B2	1094.3±5.1	11.9±0.29	1.26±0.13	0.72±0.25
B3	1102.6±3.4	11.9±0.75	1.25±0.22	1.1±1.2

Maximum SD of 1.20. SD: Standard deviation

After taking 1 cm² from different places of each patch, a good mixing of drug and polymeric materials was done, and the content uniformity test showed a small range of 1.19–1.27 of all patches with a standard deviation of 0.13–0.49. While moisture content of each polymeric film showed a low water content after preparing the formula as it did not exceed 1.05% with a maximum standard deviation of 1.20.

Gentamycin release profile

The drug release of the polymeric film produced by HPMC and PVP showed a slower gentamycin release at high HPMC content, with percentage a cumulative release of < 20% after 30 min. While upon increasing the concentration of PVP, the release of the drug was burst, with more than 15% cumulative release after only 5 min. On the other hand, polymeric film prepared by HPMC and chitosan showed longer drug release, as all formulas (B1, B2, and B3) did not reach a 100% cumulative release. In addition, both a high concentration of chitosan or HPMC showed a better-controlled release compared to HPMC and chitosan (1:1), as the release was burst for the later formula. Moreover, the polymeric film prepared by 80:20 or 20:80 HPMC and chitosan resulted in not more than 70% cumulative gentamycin release.

Gentamycin diffusion profile

Gentamycin diffusion profiles showed similar drug release profiles. The polymeric films made from both HPMC/chitosan showed lower drug diffusion in 24 h due to the control of drug release. While polymeric films prepared by HPMC/PVP showed a higher % of drug diffusion as the drug release compared to the polymeric film prepared from HPMC/chitosan. Release and diffusion data are presented in Figures 1 and 2.

Figure 1.

Drug release profiles for (a) HPMC/PVP polymeric films with loaded drug, and (b) HPMC/chitosan polymeric films loaded with gentamycin. HPMC: Hydroxypropyl methylcellulose, PVP: Polyvinylpyrrolidone

Figure 2.

Drug diffusion profiles for (a) HPMC/PVP polymeric films with loaded drug, and (b) HPMC/Chitosan polymeric films loaded with gentamycin. HPMC: Hydroxypropyl methylcellulose, PVP: Polyvinylpyrrolidone

Drug matrix association

Fourier transform infrared spectroscopy (FTIR) was used to check the ability of gentamycin to associate with the polymeric matrix without any chemical interaction with the polymers to avoid any chemical Change. As shown in Figure 3, no change was detected in the FTIR Chromatograms of the polymeric film-containing drug when compared to the polymers’ FTIR used to form the polymeric films. No change was detected, as no direct interaction was detected between the drug and the polymers in the polymeric-formed film, which means that the drug was embedded and not interacted.

Figure 3.

FTIR results of gentamycin alone, HPMC alone, PVP alone, Chitosan alone, HPMC/PVP loaded polymeric films, and HPMC/Chitosan loaded polymeric films. HPMC: Hydroxypropyl methylcellulose, PVP: Polyvinylpyrrolidone, FTIR: Fourier transform infrared spectroscopy

Antibiofilm forming assay

Polymeric films containing drug with controlled release properties (<10% release within the first 5 min) was used in the biological assay testing. According to the AlamarBlue® assay results, all polymeric films were able to release enough amount from the drug to prevent the growth of the biofilm-forming S. aureus, and the maximum bacterial growth was <10%. On the other hand, polymeric formulas made from HPMC and PVP failed to prevent the biofilms formation, which could be related to S. aureus resistance. The biofilms formed were around 20% or more, while for the polymeric formula made of HPMC and Chitosan, the biofilms formed were <5%, which correspond to a good antibiofilm activity for the prepared formulas. The released gentamycin from all the selected formulas (A1, A2, B1, and B2) was able to inhibit P. aeruginosa growth, and the bacterial viability was <10%. In the same manner, both A1 and A2 formulas failed to inhibit biofilm formation for 24 h while B1 and B2 formulas were able to inhibit biofilm formation for 24 h by <10% of the formed biofilms. Biological assay results are presented in Figure 4.

Figure 4.

Antimicrobial and antibiofilm results. (a) Staphylococcus aureus results and (b) Pseudomonas aeruginosa result

DISCUSSION

Gentamycin is a broad-spectrum aminoglycoside antibiotic with a strong activity against both Gram-positive and Gram-negative biofilm-forming bacteria.[7] The topical formula of gentamycin can decrease the serious side effects caused by the drug on the human body.[8] Using palmitic acid in combination with HPMC/PVP and HPMC/chitosan enhanced drug diffusion through Franz cells.[14] HPMC has successfully been used to control the release of a topical antibiotic formula with clindamycin.[15] PVP was used previously with antibiofilm metabolite in a microneedle formula to target skin infection, which prevailed and was found to be a successful formula to target biofilm-forming bacteria.[16] While chitosan has been used before with different antimicrobial agents to target different acne-causing microbial infections to control drug release and to add a synergistic effect as chitosan has antimicrobial activity.[16] All formulas including HPMC/PVP, and HPMC/Chitosan have been found to exert a sustained release behavior especially when HPMC and chitosan are used in a high ratio. Formulas A1 and A2 were found to control the release for 24 h, while formulas B1 and B2 were found to control the release for more than 24 h, as the cumulative release % was <70%. On the other hand, the rest of the formulas showed a burst release, as more than 15% of gentamycin was released in the first 5 min. Gentamycin as a drug is known to be used topically for skin infection with 0.1% concentration.[17] Gentamycin as an antibiotic was able to diffuse systemic circulation through the skin using a permeation enhancer to avoid its severe side effect. Thus, a longer duration of treatment using gentamycin can be considered to control drug release and avoid serious side effects such as sensorineural hearing loss.[17] All formulas showed that in the presence of palmitic acid, as a permeation enhancer, the drug diffusion was better and controlled by the drug released from the polymeric film. Formulas A1, A2, B1, and B2 were selected for characterization through scanning electron microscopy biological testing against S. aureus and P. aeruginosa for their ability to control the release of gentamycin form the polymeric films. The formula containing HPMC and chitosan showed more homogeneity compared to the formula made of HPMC and PVP.[16] The homogeneity of the transdermal patch has a great effect on polymeric film compliance for the patient and its ability to control the release of the loaded drug. According to the biological assay results of AlmarBlue® and Planktonic assay solution, gentamycin loaded in a polymeric film of HPMC/Chitosan was found to exert a stronger activity than the drug-loaded to HPMC/PVP polymeric film after 24 h. This could be resulting from the slower release of gentamycin in HPMC/Chitosan polymeric film, as more than 30% of the drug is still in the polymeric film.[16]

CONCLUSION

Gentamycin was successfully formulated as a transdermal polymeric patch after using HPMC, PVP, and Chitosan polymers. A high ratio of chitosan or HPMC in different formulas was found to exert the longest drug release, with <70% drug released in 24 h. The controlled release formulas have shown no effect of the antimicrobial, as it was able to inhibit both biofilms forming S. aureus, and P. aeruginosa after 24 h of incubation. Further investigation of drug diffusion through skin needs to be accomplished to study in vivo behavior of the drug.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.