May 2021 in Institute for Research and Community Services Universitas Muhammadiyah Palangkaraya
Method Development and Characterization of Liposomal Formulation of Isotretinoin
Abstract
This study aims to develop a liposomal drug delivery system of isotretinoin, an acne drug-using spray drying, as a cost-effective and time-effective technique. The liposomal formulation was prepared by using spray drying three different strategies were adopted suspension spray drying (SSD), thin-film hydration and spray drying (TFHSD), and emulsion spray drying (ESD). Isotretinoin was 99 bound with lipid, so lipids hydrogenated soy phosphatidylcholine (HSPC), distearoyl phosphatidylglycerol (DSPG), and cholesterol were selected for the formulation development. The HSPC, DSPG, cholesterol, and isotretinoin were taken in the ratio 4 1 0.16 3.1 mmol. In vitro drug release studies, microscopy, drug content, and related substance characterizations were done to formulate each strategy of spray drying prepared dry liposomes of isotretinoin. Results were compared with the USP monograph of isotretinoin. It was revealed that isotretinoin's liposomal formulation using ESD was having drug release according to the USP limits. Drug content was also according to the USP requirement; no free drug crystals were found in microscopy, multivesicular vesicles were found in shape, a particle size of up 60 was found. The ESD technique was a successful, time-effective, and cost-effective technique for preparing a liposomal drug delivery system for isotretinoin.
Main Text
INTRODUCTION
Acne vulgaris is awidespread skin disorder among adolescents, and it affects 90% of theindividual at some point in their life1. Its chroniccondition leads to an increase in sebum production, resulting in keratinizationchanges, inflammation under hair follicles by Propionibacterium acnes.Acne persists for a very long time with depressing consequences in adults2,3. Acne is commonlytreated using benzoyl peroxide, topical antibiotics (erythromycin andclindamycin), and topical retinoid (tretinoin, isotretinoin, retinaldehyde, andretinoyl β-glucuronide)4.
Isotretinoin is acrystalline yellowish-orange powder with a faint odor5. The isotretinoinpowder is soluble in chloroform and methylene chloride and sparingly soluble inwater6. Isotretinoinincites apoptosis (cell death) in different parts of the body cells. Apoptosismay induce hippocampus cells, hypothalamic cells, meibomian gland, and vital inacne therapy in sebaceous gland cells3,7,8. Isotretinoin hasmodest interaction for retinoid X receptors (RXR) and retinoic acid receptors(RAR) but might transform intracellularly to metabolites that mimic an agonistRXR nuclear receptors and RAR9. Isotretinoin isthe exclusive acne drug available which influences all vital pathogenic actionin acne; it differentiates from antibiotics treatments and considers itstherapeutic efficacy in severe, nodulocystic cases. The repercussions ofisotretinoin on sebum output can be short-term, or remission of the disease canbe absolute and for an extended period10.
Liposomes are smallartificial vesicles of the spherical shape created from cholesterol andnatural, nontoxic phospholipids11,12. Due to their sizeand hydrophobic and hydrophilic character (besides biocompatibility), liposomesare promising systems for drug delivery. Liposomes are extensively used ascarriers for numerous molecules in the cosmetic and pharmaceutical industries13,14.
A liposomal drugdelivery system was developed for isotretinoin using the spray drying technique15. Spray-drying usinga two-fluid nozzle was tested to dry liposome preparations, i.e., liposomescomposed of hydrogenated soy phosphatidylcholine (HSPC), distearoylphosphatidylglycerol (DSPG), cholesterol, and isotretinoin. Isotretinoin otherproducts are available in the market, such as isotretinoin suspension capsuleswhich are expensive16. Isotretinoin showsstability problems to overcome these problems and to prepare a cost-effectiveproduct to dry liposome of isotretinoin was prepared using spray dryingtechnique17. Based on thisbackground, this study aims to develop liposomal preparation of isotretinoinfor acne by using the spray drying technique.
MATERIALS AND METHODS
Materials
Isotretinoin was obtained from SunPharmaceuticals, Gurgaon, Haryana, India. All solvents (distilled water,methanol, and chloroform) were purchased from CDH (Central Drug House Pvt. Ltd,New Delhi, India). Other ingredients include borate buffer, HSPC, DSPG,cholesterol, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT),mannitol, propyl gallate, and phosphate buffer. The instruments used includethe UV spectrophotometer (Shimadzu), mini spray dryer B-290 (Buchi), EclipseE600POL microscope (Nikon), differential scanning calorimeter (DSC, Shimadzu),rotary evaporator, high-pressure homogenizer (HPH), and dissolution testapparatus type II (paddle).
Methods
Physical characterization of isotretinoin
Solubility – The saturation solubility studiesof class II model drug-like isotretinoin were conducted as per BiopharmaceuticsClassification System (BCS) guidelines18. An excess amount of isotretinoinwas placed in pH borate buffer at room temperature and stirred until saturationpoint. After 24 hours, the sample was filtered, suitably diluted, and absorbancewas taken at 346 nm against pH 8 borate buffer as blank on UVspectrophotometer. The excipient and drug solubility were observed in thesolvent, i.e., water, methanol, and chloroform, to optimize the ratio ofsolvent combination, i.e., chloroform : methanol (1 : 1) and chloroform :methanol (2 : 1).
Quantitative estimation of isotretinoin – The UV spectrophotometric methodwas used to estimate the drug because the method was simple, economical, andgives reproducible results within acceptable limits. The double beam UVspectrophotometer was used for analysis19. Isotretinoin was dissolved inborate buffer pH 8.0 containing 0.5% cetrimide. The solution was scanned formaximum absorbance in a UV double beam spectrophotometer in the range from 200to 400 nm, using the respective solution as a blank.
Selection of lipids – Optimization batches wereprepared to optimize the number of lipids. Initially, the quantities were takenin 2 : 1 mmol of HSPC and cholesterol, respectively. The Amount of DSPG waskept constant at 0.16 mmol. Tests were carried out on three batches with variationsin the number of HSPCs, as shown in Table I.
Table I. Optimization of lipidsuse for formulation development of different batches
Excipients were dissolved in thesolvent and then spray-dried by mini spray dryer B-290 with an inlettemperature of 70°C, the outlet temperature of 42°C, aspirator rate was kept at70%, nitrogen pressure was at 2 kg/cm2, and pump rate kept at 5%.The spray-dried powder was collected and dispersed in an appropriate amount ofwater and then observed under a microscope using a 50X lens.
Drug loading – Pre-optimization studies ofisotretinoin liposomal formulation were tried to formulate along with thelipids, HSPC, DSPG, and cholesterol to optimize the drug loading for theisotretinoin liposome formulation. A review of the literature revealed thatthese were the lipids that can be used in formulation development. Thus, forselection of the range, various pre-optimization trials were done with theexact quantities of lipids but with the increasing quantity of isotretinoin20, as shown in Table II.
Table II. Pre-optimizationtrials of various batches
All ingredients were dissolved inchloroform : methanol (2 : 1) solvent and spray-dried at the same parametersused earlier in this study. Batches were manufactured with isotretinoinquantities of 0.25 mmol to 3.1 mmol to check the solubility of the drug in agiven excipient. Resulted in dried drug lipid dry powder was dispersed inphosphate buffer pH 6.8 and observed for multivesicular vesicles and free drugcrystals using a microscope with a 50X lens.
Differential scanning calorimetry – Differential scanning calorimetryexperiments were performed with DSC. Sample of isotretinoin and spray driedliposomal formulation containing isotretinoin and lipids (HSPC, DSPG, andcholesterol) were submitted to DSC analysis. The analysis was performed on 5 mgsamples sealed in standard aluminum pans. Thermograms were obtained at ascanning rate of 10°C/minutes. Each sample was scanned between 0°C to 200°C.The temperature of maximal heat capacity was defined as the phase transitiontemperature21.
Preparation of liposomal formulation of isotretinoin
Isotretinoin liposomes were preparedby using a spray drying technique. The spray drying technique was employedthrough three different strategies, i.e., suspension spray drying (SSD), thin-filmhydration and spray drying (TFHSD), and emulsion spray drying (ESD).
Suspension spray drying
The solution of suspension spraydrying was prepared by dissolving HSPC, cholesterol, DSPG, and isotretinoin inthe ratio of 4 : 1 : 0.16 : 3.1 mol, respectively. These ingredients weredissolved in chloroform : methanol (2 : 1) solvent. The DSPG was dissolved inthe solvent in the presence of heat 60°C to prevent aggregates formation.Ingredients other than DSPG are mixed with the swirling of the solution22. The suspension was spray-driedwith a mini spray dryer B-290 combined with inert loop B-295 at the followingparameter: inlet temperature 60-80°C; outlet temperature 38-45°C; aspiratorrate at 50-70%; pump rate at 5-10%; and nitrogen pressure 2 kg/cm2.The dried powder so retained from the spray drier was characterized further.The batch was named SSD 1.
Thin-film hydration and spray drying
The TFHSD technique's objective wasto overcome the hygroscopicity problem that was coming in the previous SSD23. The TFHSD was conducted in severalsteps.
Preparation of solution – The solution for dispersion spraydrying was prepared with the same composition and according to the optimized amount.This was done in the solution spray drying method. Antioxidants BHA and BHT wasadded within allowed daily intake limits.
Thin-film formation using rotary evaporator – Thin-film of the solutioncontaining formulation was the suitable organic solution formed using therotary evaporator at the following parameters: bath temperature 450°C; RPM 30;vacuum 250-500 mm of Hg for 1 hour24.
Hydration of thin-film with Buffer containing5% mannitol – The hydrationof the thin-film was prepared using phosphate buffers pH 6.8 and 5% mannitol init. The hydration was carried out for 1-2 hours. The appropriate amount of thebuffer was put in the rotary evaporator vessel over the thin-film, so theformed process started again with temperature 50-55°C, and vacuum was applied.If a problem occurs in hydration, sonication was performed to recollect thetotal amount of material24. The above suspension was passedthrough the HPH to reduce the size of the particles in the suspension.
Spray drying – The prepared suspension soobtained from HPH was then spray-dried using mini spray dryer B-290 with thefollowing parameters: aspirator rate 70%; inlet temperature 80°C; outlettemperature 52°C; and pump rate 5%. The spray drying was performed using air aswell as nitrogen. The dried powder was collected and was analyzed. Thistechnique was employed to overcome the hygroscopicity25. Only related substancecharacterization was done of TFHSD. The batch was named TFHSD 1.
Emulsion spray drying
Emulsion spray drying was thetechnique in which emulsion was prepared by emulsifying organic solventcontaining drug and lipids with water containing mannitol using high-speedhomogenization25. The ESD was performed in severalsteps.
Preparation of the organic and aqueousphases – The organicphase and aqueous phase ratios were kept at 70% and 30%, respectively. Theorganic solution was prepared with the optimized quantities of the Excipientand the drug. The composition used was HSPC : cholesterol : DSPG : isotretinoin(4 : 1 : 0.16 : 3.1 mol, respectively), BHA, BHT, and propyl gallate (within alloweddaily intake limits). These all were mixed in the solvent of chloroform andmethanol (2 : 1), making 5% w/w solution, and then processed further. Mannitol(2.5/5 times of drug quantity) was dissolved in a suitable amount of distilledwater.
High-speed homogenization – The aqueous solution was addeddrop-wise to an organic solution under high-speed homogenization for speed upto 18000 RPM, resulting in an emulsion26. The homogenization was done for 5minutes with breaks for 10 seconds after 2, 3, and 4 minutes in between toavoid overheating due to heat generation.
Spray drying – The prepared emulsion was thenspray dried using the following parameters: inlet temperature 70°C; outlettemperature 42°C; aspirator rate 70%; pump rate 5%; nitrogen pressure 2 kg/cm2;and humidity of room 55-60%. The spray-dried powder was collected and filled inthe hard gelatin capsules, and analyzed further. The batch was named ESD 1.
Physical characterization of isotretinoin liposomal formulation
Drug content – As much as 10 mg of spray-driedpowder was dissolved in 100 mL of solvent, i.e., chloroform : methanol (2 : 1)in a 100 mL volumetric flask. As much as 2 mL was taken from this solution anddiluted up to 10 mL with the solvent in a 10 mL volumetric flask. Theabsorbance of the resulting solution was measured at the maximum at 346 nmusing a UV spectrophotometer27. The linearity equation obtainedfrom the calibration curve was used to estimate isotretinoin in the liposomalformulations. Results were compared with the USP monograph limits.
In vitro release studies – The release rate of 40 mg capsuleisotretinoin liposomal formulation was determined using type II dissolutiontest apparatus. The dissolution test was carried out using 900 mL of boratebuffers pH 8 with pancreatin at 37±0.5°C and 100 RPM using a 40-mesh basket.Sample no. 108 (10 mL) was withdrawn from the dissolution apparatus andreplaced with a fresh medium. Sample no. 109 was filtered through a 0.45 μmmembrane and diluted to a suitable solvent. The absorbance of samples wasmeasured at 346 nm using a UV spectrophotometer, and cumulative drug releasewas calculated28. Sample no. 110 drug release datawere analyzed using PCP Disso software version 2.08. The dissolution profile ofall the batches was fitted to zero-order, first-order, Higuchi, Hixson-Crowell,as well as Korsmeyer and Peppas to ascertain the kinetic modeling of drugrelease, and the model with the highest correlation coefficient was consideredto be the best mode. Results were compared with the USP monograph limits.
Microscopy – Liposomal dispersion was preparedby dispersing spray-dried formulation in phosphate buffer pH 6.8 and wasobserved under 50X lens under Polarizing microscope. The microscopy revealedthe particle size, shape of vesicles (regular or irregular), and free drugcrystals29. The 50X resolution lens was usedfor the particle size analysis. In a microscope, 1 unit of the scale present onthe lens was taken as 2µ. The required amount of blend was reconstituted withthe necessary amount of water, and then the slide was prepared and observedunder a microscope.
Related substances (detection of impurity) – This characterization was done to checkimpurities in the prepared liposomal formulation of isotretinoin—5,6epoxy-13-cis retinoic acid, tretinoin, and others unknown. Some impurities wereobserved with the related substance test of the liposomal formulation30. The results were compared with theUSP monograph limits.
RESULTS AND DISCUSSION
Physical characterization of isotretinoin
Solubility studies – The isotretinoin was found to beyellowish-orange, crystalline powder with a faint odor that resembles vitaminA. The drug was insoluble in water, whereas it was soluble in the organicsolvent, thereby suggesting it was lipophilic. Chloroform : methanol (2 : 1respectively) was taken for the formulation as solvents. Isotretinoin exhibitssolubility across the borate buffer pH 8. Therefore, it could be classified asa “less soluble” material. A review of the literature revealed that the drugexhibits high permeability. According to BCS classification, if the highestdose of the drug was insoluble in aqueous media and exhibited highpermeability, it was called a BCS class II drug18. Therefore, the isotretinoin fallsunder BCS class II.
Selection of lipids – Excipient for isotretinoinliposomal formulation was selected based on maximum solubility, in whichisotretinoin was found to be a lipophilic drug. Batches were prepared andobserved under the microscope 50X lens for free drug crystals, and the shapeand size of vesicles formed to determine the excipient ratios used. Batch no. 1had shown the presence of free drug crystals while vesicles were irregular inshape. Batch no. 2 had also shown the presence of free drug crystals, andvesicles were also irregular in shape and size. Batch no. 3 had not shown anyfree drug crystals, but multivesicular structures were regular in round shapes.As observed, batch 3 excipient ratios were selected for the preparation of theliposomal formulation31. Clear vesicular structures with nofree drug crystals found with HSPC : cholesterol 4 : 1. Therefore, the 4 : 1ratio was found to be sufficient.
Drug loading – Isotretinoin quantity of 3.1 molresulted in powder blend, which contains 40 mg drug in 200 mg blend.Hygroscopicity was observed, and for the collection of spray-dried powder, RHwas maintained below 25%. The collected powder was filled in 00 sizes hard gelatincapsule with a suitable amount. Further increase in drug quantity leads to freedrug crystals. The drug loading of various batches in different quantities waspresented in Table III.
Table III. Microscopic observation for drugloading
Differential scanning calorimetry – Differential scanning calorimetry study wascarried out for isotretinoin and drug-loaded dry liposome’s containing lipids(HSPC, DSPG, and cholesterol). The DSC thermograms of isotretinoin anddrug-loaded dry liposomes were illustrated in Figures 1 and 2, respectively. The DSC thermogramof isotretinoin showed endothermic at 181.73°C. It proves that the isotretinoinwas pure32.
The DSC thermogram of drug-loadeddry liposomes with lipids showed endothermic at 76.58°C. The DSC thermogram ofisotretinoin-loaded liposomes composed of HSPC, DSPG, cholesterol, andisotretinoin in the ratio of 4 : 1 : 0.16 : 3.1, respectively, showed thedisappearance of melting endothermic of isotretinoin and major endothermic wasobserved at 76.58°C. The absence of the melting endothermic of isotretinoinsuggested significant interaction of isotretinoin with lipids leading toenhanced entrapment of the drug33. The isotretinoin was dissolved inlipids. The incorporated isotretinoin was associated with lipids and interactedto a large extends with them. The DSC results of liposome’s suggested enhancedentrapment efficiency of isotretinoin in a lipid bilayer.
Microscopy – Microscopy of batches was done to observe particle size, shape, andfree drug crystals. The particle size multivesicular vesicles of sizes 2 to 60µwere observed. Figures3a and 3b of SSD 1 microscopy showed that multivesicularvesicles of drug solubilized lipids were found. Free drug crystals were notfound. The drug was solubilized entirely in lipids. In Figure 3c of TFHSD 1, microscopy-free drug crystals werefound, and the size and shape of the vesicles were also irregular. Thus, thestrategy was dropped, but beneficial points were taken into considerations fromthis strategy, such as this strategy was successfully able to removehygroscopicity. In Figure 3d of ESD 1, microscopy large lipidvesicles in the aqueous medium were found. No free drug crystals were found.
In SSD 1, no free drug crystals were found, and the vesicles were alsoin regular shape and size, which states that the drug was utterly solubilizedin lipids34. In ESD 1, large lipid vesicleswere found, and no free drug crystals were found. Thus, both these strategiespass the microscopy test. In TFHSD 1, free drug crystals were found, and thevesicles were irregular in shape; this strategy fails the microscopy test.
Figure 1. Differential scanning calorimetrythermogram of isotretinoin
Figure 2. Differential scanning calorimetrythermogram of dry liposomes of isotretinoin and lipids
a b
c d
Figure 3. The SEM images of SSD 1 (a and b),TFHSD 1 (c), and ESD 1 (d) taken at 20,000x magnification
Physical characterization of isotretinoin liposomal formulation
Drug content – The formulation should containnot less than 90.0% and not more than 110.0% of the labeled amount of drugaccording to the USP monograph [120]. The triplicate batches were prepared forevery strategy, and the assay was also done in triplicate average best assayresults35. Batch SSD 1 had shown drug content96.6%, while batch ESD 1 had shown 91.1% of drug content. The assayed drugcontent in formulations ranges from 90.0 % to 110.0 %, which were under the USPlimit.
In vitro drug release – The in vitro drug release studies were performed in boratebuffer with pancreatin to determine the release of the drug. The dissolutionprofiles of all the formulations were determined and shown in Figure 4. The results were analyzed by plotting cumulativedrug release/time. The dissolution was done in low intensity of light as thedrug was light-sensitive. The dissolution was done in triplicates of eachstrategy.
The average best results were shownin Figure 4—the dissolution profile of SSD 1and ESD 1. According to the USP monograph, 70% of the drug should be releasedin 90 minutes. Dissolution of SSD 1 batch slow-release, which did not fulfillthe USP requirement as there was 61% release in 90 minutes. Dissolution of ESD1 batch 95% release in 90 minutes fulfills USP requirement and shows immediaterelease action36.
Figure 4. In vitro drug release profile
Related substances – The related substance analysis wasdone to detect impurity in the formulation, which was compared with the USPlimits37. The analysis was done intriplicates of each batch, and the best results were given in Table IV. The analysis revealed that the impurities ofbatch SSD 1 and ESD 1 were according to the USP limits. The impurities of batchTHFSD 1 (using nitrogen; 1N and using air; 1A) were not found out of the limitsof USP.
Table IV. Relatedsubstance (impurity testing) data
CONCLUSION
Dry liposomes of isotretinoin were successfully preparedusing three different strategies of spray drying, i.e., SSD, TFHSD, and ESD.The formulation of each respective strategy was analyzed. Related substance(impurity study) revealed that the impurities of SSD and ESD were under the USPlimits, whereas the impurities of TFHSD were out of USP limits, so theformulation of two strategies SSD and ESD, were further analyzed. Therefore, aliposomal formulation of isotretinoin through ESD was found successful, time-effective,and cost-effective in comparison with the other two strategies.
ACKNOWLEDGMENT
The authors are thankful to Professor Teerapol Srychana,Prince of Songkla University, Hatyai, Thailand, for motivation, guiding, andchecking the English language of the paper.
AUTHORS’ CONTRIBUTION
Md. Iftekhar Ahmad: Conceptualization, Project administration,Supervision, Investigation; Punet Kumar: Formal Analysis, Fundingacquisition, Data curation, Methodology, Resources, Software; Sangam Singh:Validation, Visualization, Writing - original draft; Nitin Kumar:Writing - review & editing.
DATA AVAILABILITY
All data are available from the authors.
CONFLICT OF INTEREST
The authors declare no conflict of interest or otherwise.
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Abstract
Main Text
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
CONCLUSION
ACKNOWLEDGMENT
AUTHORS’ CONTRIBUTION
DATA AVAILABILITY
CONFLICT OF INTEREST
REFERENCES