Liposomal preparations of benzoyl peroxide for the treatment of acne

LIPOSOMAL PREPARATIONS OF BENZOYL PEROXIDE FOR THE TREATMENT OF ACNE ONG SHWU YNG B Sc Pharm (Hons.), NUS A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF SCIENCE DEPARTMENT OF PHARMACY NATIONAL UNIVERSITY OF SINGAPORE 2005 Acknowledgements This project would not have been successfully completed without the support and encouragement of the following people: My supervisor and mentor, Associate Professor Lim Lee Yong, for her dedicated supervision and guidance and for her invaluable advice during the course of this project; Consulting Associate Professor Go Mei Lin who has offered numerous advice and suggestions; Laboratory officers Wong Lai Peng, Matthew Tham, Ng Sek Eng, and Mr Tang Chong Wing who have helped me with obtaining my materials and troubleshooting equipment faults; Research students from my team Mo Yun, Cheng Weiqiang, Huang Min, Lim Siok Lam, Han Yi, Ren Yu Peng, Zhang Wenxia, Jiang Dahai, and research assistant Erna for their advice and facilitation in my project Heartfelt gratitude is also extended to my family and friends for their support and encouragement throughout the course of this project Ong Shwu Yng 2005 i Table of Contents Introduction Hypothesis Materials Methods 4.1 Preparation of liposomes and drug encapsulation 4.2 Characterization of liposomes 4.2.1 High Performance Liquid Chromatography (HPLC) 4.2.2 Encapsulation efficiency of liposomes 4.2.3 In-vitro drug release 4.2.4 Size, size distribution and polydispersity 4.2.5 Stability of liposomes 4.2.6 Morphology 4.3 In-vitro inhibition of Propionibacterium acnes (P acnes) 4.3.1 Culture of P acnes 4.3.2 Antibacterial assay 4.4 Statistical analysis Results and Discussion 5.1 Preliminary experiment 5.2 Effect of drug loading 5.2.1 Characteristics of liposomes 5.2.2 Microbiological activity ii 5.3 Effect of storage 5.3.1 Characteristics of liposomes 5.3.2 Microbiological activity Conclusion Future Work References Appendices iii Summary Benzoyl peroxide (BPO) is used extensively for the treatment of acne but is known to induce concentration-related skin irritation We postulate that this side effect may be prevented by using BPO encapsulated in liposomes The objectives of this study were to develop, optimize and characterize liposomal preparations of BPO to determine its antibacterial efficacy and storage stability BPO-loaded liposomes having mean size ranging from 250 to 900 nm were successfully prepared using a combination of cholesterol and phosphatidylcholine Sonication was necessary to obtain the smaller sized liposomes The residual BPO content in the liposomes was dependent on the pressures applied during the drying down and hydration of lipids Applying a low pressure during drying down led to a rapid evaporation of the organic solvent, and resulted in a substantial drug loss Processes involved in extracting the drug from the liposomes for the determination of drug loading, e.g method and duration of agitation, and method of filtration, did not affect the BPO content in the liposomes BPO-loaded liposomes prepared with operating pressures of 450 atm during solvent evaporation and 150 atm during lipid hydration yielded a high encapsulation efficiency of >90% Sustained release of the encapsulated BPO was obtained upon dilution with an aqueous medium at pH 7.4, with less than 40% of the drug load released in 24 h Smaller liposomes of 350 nm, prepared by prolonging the duration of sonication, were found to have slower rates of drug release due to their propensity to form aggregates iv Liposomes containing BPO concentrations ranging from 12.5 to 100.0 mg/ml were prepared for the study of its antibacterial activity against P acnes Upon storage for weeks at 4°C, the mean size of the liposomes increased from 800 nm to more than 2000 nm while those stored for weeks at room temperature decreased in size from 800 nm to 400 nm or less To obtain more stable liposomes, the phosphatidylcholine:cholesterol mole ratio was increased from 3:1 to 1:1 These liposomes showed much smaller changes in mean size upon storage However, the BPO encapsulation efficiency was reduced from 74 - 83% to 60 - 65% after weeks of storage at 4°C and room temperature This reduction was due to the diffusion of BPO out of the liposomes over time, as evident from the results of the in vitro drug release studies, in which only about 50% of the BPO was released after 24 h The antibacterial efficacy of the BPO-loaded liposomes, determined by measuring the zone of inhibition produced against P acnes, was not affected by the increase in the cholesterol content of the liposomes However, the antibacterial efficacy of the liposomes was increased after storage, as evident from the bigger zones of inhibition produced by liposomes stored for weeks at either 4°C or room temperature In contrast, the control BPO solutions produced comparable zones of inhibition before and after storage From these results, it is clear that BPO retained its antibacterial activity against P acnes when encapsulated in liposomes Key Words: Nano-liposomes; Acne Treatment; Benzoyl Peroxide; Topical Drug Delivery; Antibacterial Activity v List of Tables Table Description of liposomes according to particle size Table Samples used for the antibacterial assay against P acnes Table Effect of sonication duration on the size distribution of liposomes Table BPO encapsulation efficiency (EE, %) of liposomes prepared with different drying and hydration pressures EE was calculated from the BPO contents in the liposome dispersion before (precentrifugation sample) and after centrifugation (pellet and supernatant) (Values represent Mean ± SD, n = 3) Table Loss of BPO due to rotary evaporation and post-evaporation processing of the BPO-lipid mixtures Table Effects of the method and duration of agitation on the recovery of BPO (%) from BPO solutions and BPO-loaded liposomes Recovery was determined by expressing the BPO content as a percentage of the BPO load (~950 µg) obtained post-evaporation in the solution and liposomes samples (Mean ± SD, n = 3) Table Effect of filtration on the recovery of BPO from BPO-loaded liposomes and BPO solutions Recovery was calculated as a percent of the initial BPO load (~950 µg) obtained post evaporation (Mean ± SD, n = 3) Table The effect of pressure applied during rotary evaporation on the loss of BPO from the liposomes Recovery was calculated based on the initial BPO load of ~1800 µg (Mean ± SD, n = 3) Table Effect of BPO concentrations: mg/ml (BL0), 12.5 mg/ml (B12.5), 25 mg/ml (B25), 50 mg/ml (B50), and 100 mg/ml (B100) on the size distribution of liposomes prepared with the lipids PC: CH in the ratio of 3:1 (L31) and 1:1 (L11), respectively Values represent mean size ± SD, n = Table 10 BPO encapsulation efficiency (EE, %) of liposomes prepared with PC:CH mole ratios of 3:1 or 1:1, and with BPO loading concentrations of 12.5, 25, 50 or 100 mg/ml EE was calculated from the BPO contents in the liposome dispersion before (precentrifugation sample) and after centrifugation (pellet and supernatant) Values represent Mean ± SD, n = vi Table 11 Effect of storage on the sizes of BPO-loaded liposomes prepared with PC:CH ratio of 3:1 and 1:1 Liposomes were stored for weeks at 4°C and at room temperature Values represent mean ± SD, n = Table 12 BPO encapsulation efficiency (EE, %) of liposomes prepared with PC: CH in the mole ratio of 1:1 and BPO concentrations of 12.5 mg/ml (L11B12.5), 25 mg/ml (L11B25), 50 mg/ml (L11B50), 100mg/ml (L11B100) after weeks of storage in the fridge and under room temperature EE was calculated from the BPO contents in the liposome dispersion before (precentrifugation sample) and after centrifugation (pellet and supernatant) (Mean ± SD, n = 3) vii List of Figures Figure Flow chart of liposome preparation and determination of BPO content encapsulation efficiency Figure 2.1 Determination of the zone of inhibition for each cup on the agar plate Figure 2.2 Positions of cups A to F on the agar plate Figure In vitro release profiles of BPO from liposomes produced with different durations of sonication: (B-0), 10 (B-10) and 20 (B-20) Control experiments were conducted with BPO solutions in the presence (C-1) and absence (C-2) of the dialysis bag (Mean ± SD, n = 3) Figure TEM photomicrographs of liposomes prepared with 10 sonication (B10) and with 20 sonication (B-20) Liposomes were observed at magnification of 28.5K (a and b), and 11.5K (c and d) Figure Mean particle size of BPO-loaded liposomes after storage at 4°C for various period of time Batch was prepared without sonication Batches to were prepared with 100 sonication Figure TEM photomicrographs of BPO liposomes (B0) prepared with the lipids PC: CH in the ratio of 3:1 (a) and 1:1 (b) Liposomes were observed at magnification of 52.0K Figure In vitro release profiles of BPO from liposomes produced with lipids PC: CH in the ratio of 3:1 and with different BPO concentrations: 12.5 mg/ml (L31B12.5), 25 mg/ml (L31B25), 50 mg/ml (L31B50) and 100 mg/ml (L31B100) Control experiments were conducted with BPO solutions of different concentrations: 12.5 mg/ml (S0B12.5), 25 mg/ml (S0B25), 50 mg/ml (S0B50), 100 mg/ml (S0B100) (Mean ± SD, n = 3) Figure In vitro release profiles of BPO from liposomes produced with lipids PC: CH in the ratio of 1:1 and with different BPO concentrations: 12.5 mg/ml (L11B12.5), 25 mg/ml (L11B25), 50 mg/ml (L11B50) and 100 mg/ml (L11B100) Control experiments were conducted with BPO solutions of different concentrations: 12.5 mg/ml (S0B12.5), 25 mg/ml (S0B25), 50 mg/ml (S0B50), 100 mg/ml (S0B100) (Mean ± SD, n = 3) Figure Zones of inhibition against P acnes for BPO-loaded liposomes prepared with PC:CH mole ratios of 3:1 (L31B) and 1:1 (L11B), and the control BPO solution (S0B) viii Figure 10 TEM photomicrographs of liposomes prepared with lipids PC:CH in the ratio of 3:1 and BPO loading concentration of 25 mg/ml Liposomes had been stored for weeks at 4°C (a and b) and at room temperature (c and d) before TEM analysis at magnification of 28.5K (a) 15.5K (b), and 52.0K (c and d) Figure 11 In vitro release profiles of BPO from liposomes produced with different BPO concentrations and stored in the fridge (a) and under room temperature (b) for weeks: 12.5 mg/ml (L11B12.5), 25 mg/ml (L11B25), 50 mg/ml (L11B50) and 100 mg/ml (L11B100) Control experiments were conducted with BPO solutions of different concentrations: 12.5 mg/ml (S0B12.5), 25 mg/ml (S0B25), 50 mg/ml (S0B50) and 100 mg/ml (S0B100) (Mean ± SD, n = 3) Figure 12 Zones of inhibition of BPO liposomes with PC: CH at mole ratio 1:1 before storage (L11B) and after it is stored in the fridge (L11B-FG) and under room temperature (L11B-R) Figure 13 Zones of inhibition of BPO solutions before storage (S0B) and after it is stored in the fridge (S0B-FG) and under room temperature (S0B-R) ix (200-300 nm) Increasing the PC:CH mole ratio to 1:1 stabilized the liposomes, and the changes in mean size of the liposomes after storage for weeks at 4°C and ambient temperatures were much smaller Although the higher cholesterol level in the liposomes increased the BPO encapsulation efficiency to between 74 to 83%, these values were, however, lower than the EE value of >90% obtained for liposomes prepared with very low BPO content The lower encapsulation rate translated to faster rate and extent of drug from the liposomes Storage for a further weeks increased the amount of free BPO in the liposomal dispersion, with the result that more than 80% of the BPO load was released from the liposomes within h The storage temperature of 4°C and room temperature, as well as the BPO loading concentration in the liposomes, had minimal effects on the size distribution, EE, and in vitro BPO release profile of the liposomes BPO in the phospholipids liposome formulations had an antibacterial effect against P acnes For effective inhibition of P acnes, BPO concentrations of 25 mg/ml and above had to be used to prepare the liposomal samples Antibacterial efficacy was not affected by the ratio of phosphatidylcholine and cholesterol in the lipid bilayer nor the storage temperature More studies have to be conducted to confirm if BPO encapsulated in liposomes exhibit sustained release 63 Future Work In the present work, BPO encapsulated in liposomes have been successfully developed, optimized and characterized BPO liposomes have also exhibited in-vitro antibacterial activity against P acnes, the major cause of inflammation in acne Although pure BPO solutions in this study were more effective in inhibiting the bacteria, future studies will have to be conducted to determine whether the liposomal BPO is not only effective in inhibiting P acnes but also in reducing the skin irritation and itching common associated with a conventional BPO formulation The next phase of work would be to optimize the formulation so that the BPO liposome dispersions may be developed into pharmaceutically relevant products Additional work will be required to 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Chemother 32, 1204-1207 Zulli, F., and Suter, F., 1994 Preparation of small lipid nanoparticles for topical applications Proceed Intern Symp Control Rel Bioact Mater 21, 459-460 76 Appendices BPO Standard Curve 60000 y = 245.65x R = 0.9936 Peak Area 50000 40000 30000 20000 10000 0 50 100 150 200 250 Concentration Appendix BPO standard curve determined by HPLC Day 10 0.928 3.527 3.464 BPO concentration µg/ml 50 100 150 1.416 0.884 0.035 4.470 0.202 1.360 1.318 1.650 1.190 200 0.002 1.060 0.383 Appendix Coefficient of variation for BPO liposomes analyzed over days 77 [...]... scrubbing of the skin (Ghyczl et al., 1996) Scientists have made tremendous progress over the last twenty-five years in their research of the pathogenesis of acne, and improved treatment alternatives have caused the lives of many patients to change for the better Drugs currently used for the treatment of acne include benzoyl peroxide, antibiotics, retinoids and oral contraceptives Procedureoriented acne treatments... 1978) These free fatty acids stimulate the hair follicle, form the comedo, and then induce the inflammation which leads to the formation of papules and pustules (Downing et al., 1986) 2 The three important physiological factors in the pathogenesis of acne are the multiplication of P acnes, the overproduction of sebum and follicular hyperkeratinization It is widely known that the action of P acnes lipase... impact on the physical properties and biological fate of the liposomes and their entrapped substances These parameters affect the physical stability, in vivo distribution and size uniformity of the liposomal formulation The determination of drug loading provides information on drug loss during preparation, as 13 well as the percent ratio of entrapped to free drug present in the formulation The latter... in the mid 1990s, followed quickly by formulations of tretinoin and tazarotene, which were less irritating, yet more effective in the treatment of acne Topical retinoids, either alone or in combination with other acne medications (e.g tretinoin and oral antibiotics), have since been promoted as an effective treatment for inflammatory acne They are also recommended for maintenance therapy after the. .. said, the long-term use of antibiotics for acne treatment continues to raise concerns regarding the development of colonization with potential pathogens and bacterial resistance Oral contraceptive is another accepted therapeutic modality for the treatment of acne in women All combination oral contraceptive pills have the potential to improve acne, and they work by increasing the sex hormone-binding... with the aqueous phase The detergent associates with the lipid molecules and functions to screen the hydrophobic portions of the molecules from water Micelles which form from this association are composed of several hundred component molecules, their shape and size depending on the involvement of other lipids and the chemical nature of the detergent After the detergent 10 is subsequently removed from the. .. minimize the leaching out of an encapsulated water-soluble drug, cholesterol is usually included in the formula In addition, the incorporation of cholesterol into liposomal bilayers also decreases the rotational freedom of the lipid hydrocarbon chains (De Gier et al., 1968) Liposomal formulations are good for topical application because they can spread excellently to form depots of active ingredients in the. .. 1997), liposomal formulations are indeed able to reduce the side effects of the various drugs encapsulated and are able to enhance the accumulation of the drugs at the administration site Despite the scientific interest, the correlation between the liposome preparation method and the characteristics of the BPO-loaded liposomes remain poorly understood (Patel et al., 2001) Depending on the methods of preparation,... 1965) Therefore, P acnes is considered to play an important role in acne development by secreting inflammation-inducing factors 1.3 Treatment of acne The past 25 years have brought about significant changes in the treatment of acne Topical retinoids, which are anti-inflammatory and comedolytic, became the mainstream acne treatment in the early 1980s, but problems with skin irritation limited their... microscopy (Toyoda and Morohashi, 2001) The lesion results in micro-comedo, which then leads to closed or open comedone Inflammatory lesions begin to form when the proliferation of P acnes causes the secretion of leukocyte chemotatic factors, which progresses to the infiltration of leukocytes into the hair follicle Thereafter, the contents of the hair follicle flow into the dermis to initiate inflammation ... Efficiency D1 Diameter of the zone of inhibition d The distance from the edge of the cup to the edge of the zone of inhibition x Introduction 1.1 Acne Acne is an inflammatory disease of the sebaceous... cup was measured, then subtracted from the zone of inhibition (D1) and divided by to obtain the value of d (i.e the distance from the edge of the cup to the edge of the zone of inhibition) (Figure... the lives of many patients to change for the better Drugs currently used for the treatment of acne include benzoyl peroxide, antibiotics, retinoids and oral contraceptives Procedureoriented acne