Formulation and Evaluation of Anti-Acne Niosomal Gel using Retinol and Lincomycin Combination

Acne vulgaris is one of the most prevalent chronic dermatological conditions, affecting a large proportion of adolescents and a significant number of adults worldwide. Although often considered a cosmetic concern, acne can have profound psychological and social consequences, impacting quality of life. The pathogenesis of acne is complex and involves multiple interrelated factors, including excessive sebum production, abnormal keratinization of hair follicles, proliferation of Cutibacterium acnes, and inflammation within the pilosebaceous unit¹.

Topical therapy remains the first-line approach for mild to moderate acne due to its localized action and reduced systemic side effects. Among topical agents, retinoids such as retinol and its derivatives play a central role. These compounds, structurally related to vitamin A, regulate epithelial cell turnover, prevent comedone formation, and exhibit anti-inflammatory effects. Despite their effectiveness, retinoids are often associated with skin irritation, dryness, and poor stability when exposed to light and oxygen, which can limit patient adherence².

In addition to retinoids, topical antibiotics are frequently used to control bacterial proliferation and inflammation. Lincomycin, a lincosamide antibiotic, has demonstrated activity against Gram-positive organisms, including C. acnes, and can reduce inflammatory lesions when applied topically. However, prolonged use of antibiotics alone may lead to bacterial resistance and reduced efficacy, emphasizing the importance of combination therapy³. The integration of retinoids with antibiotics offers a synergistic approach, addressing both comedogenesis and microbial involvement in acne pathophysiology.

Despite the therapeutic advantages of these agents, conventional topical formulations often suffer from limited skin penetration, rapid drug degradation, and suboptimal drug retention at the target site. To overcome these challenges, novel drug delivery systems such as niosomes have gained considerable attention. Niosomes are non-ionic surfactant-based vesicular carriers capable of encapsulating both hydrophilic and lipophilic drugs. These vesicles enhance drug stability, improve permeation through the stratum corneum, and provide sustained release, thereby increasing therapeutic efficacy while minimizing adverse effects⁴.

Niosomal gels, in particular, combine the advantages of vesicular systems with the convenience of topical gel formulations. They offer improved patient compliance due to ease of application, non-greasy texture, and enhanced drug residence time on the skin. Several studies have demonstrated that niosomal formulations of anti-acne agents result in better skin deposition and controlled drug release compared to conventional preparations^4,5. Furthermore, combining two active agents within a single niosomal system may provide a more effective and targeted therapy by delivering drugs simultaneously to the affected site.

Based on these considerations, the present study focuses on the formulation and evaluation of a niosomal gel containing a combination of retinol and lincomycin. This approach aims to enhance drug stability, improve skin penetration, and achieve a synergistic therapeutic effect while reducing the limitations associated with conventional formulations. Such a system has the potential to offer an improved and patient-friendly strategy for the management of acne vulgaris.

2. AIM & OBJECTIVE OF THE STUDY

2.1 Aim of the Study

The present research work is aimed at the formulation and evaluation of a niosomal gel containing a combination of retinol and lincomycin hydrochloride for enhanced topical delivery in the management of acne vulgaris.

The study focuses on improving drug stability, enhancing skin permeation, and achieving controlled drug release through a novel vesicular drug delivery system.

2.2 Objectives of the Study

To accomplish the above aim, the following specific objectives were designed:

2.2.1 Pre-formulation Studies

• To determine the physicochemical properties of retinol and lincomycin hydrochloride, including:
  • Physical appearance
  • Melting point
  • Solubility profile
  • Partition coefficient
  • λmax determination
• To evaluate drug–excipient compatibility using FTIR and other analytical techniques.

2.2.2 Formulation of Niosomes

• To prepare niosomal formulations of retinol and lincomycin hydrochloride using the ether injection method.
• To select suitable surfactants (Span and Tween series) and cholesterol for vesicle formation.

2.2.3 Optimization of Formulation Variables

• To optimize the surfactant-to-cholesterol ratio based on:
  • Entrapment efficiency
  • Particle size
  • Stability parameters

2.2.4 Evaluation of Niosomal Formulations

• To characterize the prepared niosomes for:
  • Particle size and polydispersity index (PDI)
  • Zeta potential
  • Vesicle morphology (TEM/optical microscopy)
  • Percentage entrapment efficiency

2.2.5 In Vitro Drug Release Study

• To evaluate the release profile of retinol and lincomycin from niosomal formulations.
• To compare the release behavior with that of plain drug solutions.

2.2.6 Formulation of Niosomal Gel

• To develop a carbopol-based gel system for incorporation of optimized niosomal formulations.
• To ensure appropriate pH and consistency for topical application.

2.2.7 Evaluation of Gel Formulation

• To assess the prepared gel for:
  • Physical appearance (color, homogeneity, consistency)
  • pH
  • Viscosity

2.2.8 In Vitro Permeation Study

• To study drug permeation through a semi-permeable membrane using Franz diffusion cell.
• To determine cumulative drug release and permeation kinetics.

2.2.9 Overall Performance Evaluation

• To compare the developed niosomal gel with conventional formulations in terms of:
  • Drug release
  • Skin permeation
  • Potential therapeutic effectiveness

3. MATERIAL AND METHODS

3.1 Chemicals and instrumentation

Various materials from standard suppliers were procured. Table explains the list of chemicals used throughout the study and their supplier name and location. Instrumentation list have been explained in table .

Table 1: List of chemicals

Table 2: List of instruments

3.2 Pre-formulation studies

It is an important tool for determination of physical and chemical properties of drug before incorporating in formulation development. This is the first step in rational development of dosage forms of a drug substance which gives information needed to define the nature of drug substance and provide framework for the drug combination with pharmaceutical excipients. The nature of the drug highly affects the processing parameters like method of preparation, entrapment efficiency, compatibility and pharmacokinetic response of the formulation. These are indispensable protocol for the development of safe, effective as well as stable dosage form. Thus, in order to ensure optimum condition for clinically beneficial delivery system, preformulation studies were carried out.

Various Pre-formulation parameters

1. 1. 1. Physical Description

The drugs were inspected visually.

1. 1. 2. Melting point

It is a criterion for purity as well as for identification. Capillary melting point apparatus was used to determine melting point of Retinol acid and Lincomycin. Small amount of Retinol acid was filled in capillary and temperature at which drug melted was noted down. Same procedure was repeated for Lincomycin⁶.

1. 1. 3. Fourier Transform Infrared spectroscopy

FTIR studies investigate any physicochemical interactions between components in the formulation and can therefore be applied to the selection of suitable chemically compatible excipients. FTIR spectroscopy of Retinol acid and Lincomycin were performed by using FTIR 8400S Shimaadzu⁷.

1. 1. 4. Solubility

The solubility of Retinol acid and Lincomycin were tested in various solvents such as distilled water, ethanol, methanol, chloroform and PBS pH 7.4.

1. 1. 5. Determination of ( λmax) absorption maxima (IP 2007)

A 10 ug/ml solution of Retinol acid as well as Lincomycin were prepared in methanol and distilled water respectively. This solution was further scanned in the range of 200-400 nm using UV-visible spectrophotometer.

1. 1. 6. Preparation of calibration curve

Accurately weighed 100 mg of Retinol acid was dissolved in 100 ml of methanol to get stock solution of 1000 ug/ml, from this stock solution 10 ml was withdrawn and further diluted to 100 ml with methanol to obtain 2^nd stock solution having of 100 µg/ml. 10 ml solution was taken from later stock solution and diluted to 100 ml with methanol. Aliquots of 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0 and 10.0 ml were taken in volumetric flask and volume was made upto 10 ml. Absorbances of all solutions were measured at 352 nm. Similarly weighed 100 mg of Lincomycin dissolved in 100 ml of water to get stock solution of 1000 µg/ml and same procedure was repeated as above at 210 nm.

1. 1. 7. Partition coefficient

It provides thermodynamic measure of hydrophilicity-lipophilicity balance of a chemical compound which was determined in n-octanol: water system⁸. The n-octanol: Aqueous mixture (1:1 v/v) was kept on shaker for shaking after adding the drug in it. Next day kept it on plain surface for 24 hrs till it attains equilibrium. Then both phase get separately and the concentration of drug in each phase gets measured.

logP=logconcentration of soluteOctanolconcentration of soluteAqueous

3.3 Formulation of niosomes (Tarekegn et al., 2010)

Retinol acid as well as Lincomycin loaded carrier system was prepared using ether injection method. In this method lipid was first dissolved in an organic solution which was then brought into contact with aqueous phase containing materials to be entrapped within the vesicles. In brief, surfactant i.e. (span 60, span 80, tween 60, tween 80) and cholesterol in different ratio were dissolved in 10 ml diethyl ether. Drug solution was prepared by adding drug into 10 ml phosphate buffer pH 7.4. Then dissolved surfactant/lipid were injected slowly at the rate of 0.25 ml/min through 23 gauge needle into 10 ml drug solution which is magnetically stirred continuously and maintained at 60 ^?C for 1 hr to ensure complete evaporation of solvent and to get uniform suspension of niosomes. Concentration of niosomal ingredients and process variables were optimized on the basis of size, shape, zeta potential and entrapment efficiency. Following steps were used for both drugs

1. 4. Evaluation parameters of niosomes
      1. Particle size and polydispersity index (PI) measurements

The measurements were taken by using Beckman coulter counter size analyzer at a temperature of 20 ^?C under a fixed angle of 90^?. Dispersions were diluted suitably with distilled water.

1. 1. 2. Zeta potential (Bayindir et al., 2010) 

It is a scientific term for electro-kinetic potential in vesicle systems. This is potential difference between dispersion medium and stationary layer of fluid attached to the dispersed particle. Zeta potential was measured by using flow through cell cuvette, working on the principle electrophoretic light scattering (ELS), which determines electrophoretic movement of charged particles under an applied electric field from Doppler shift of scattered light

1. 1. 3. Transmission electron microscopy (TEM)

Niosomes preparations were characterized for their shape as well as surface morphology using transmission electron microscopy. For TEM imaging, copper grids having a thin layer of carbon were loaded with T-MNLC dispersion. Sample was allowed to dry under IR lamp   and images were captured⁹.

1. 1. 4. Percentage entrapment   efficiency

Percentage of Retinol acid and Lincomycin hydrochloride entrapped in the niosomes was determined by centrifugation of formulation at 25000 rpm for half an hour at controlled temperature of 4 ^?C. Supernatant was withdrawn and measured by UV spectrophotometer at 352 nm and 202 nm respectively^10,11 (Azmin et al., 1985 and Ramchandran et al., 2010). Entrapment efficieny was calculated by using following formula¹²:

% Entrapment efficiency =entrapped drugtotal drugx100

3.4.5 In- vitro drug release study of niosomes loaded with Retinol acid and Lincomycin   

In-vitro release kinetics of Retinol acid and Lincomycin was performed using dialysis method. Incubator shaker was kept at constant temperature 37 ^?C with 100 rpm. Semi permeable cellophane membrane (previously immersed in phosphate buffer pH 7.4 for 24 hrs) was firmly stretched over the lower open end of a glass tube made watertight by rubber band (donor compartment). The tube was then immersed in a beaker containing 200 ml of phosphate buffer pH 7.4 (receptor compartment). Samples were analyzed spectrophotometrically at respective λmax¹³.

1. 5. Preparation of cellophane membrane sac

A 10 cm long portion of the cellophane membrane was made in the form of sac by folding and tying up one end of the membrane with thread, taking care to ensure that there would be no leakage of the contents from the sac. It was soaked overnight in the buffer medium.

3.5.1 Cellophane membrane set up

The wet sac was gently opened and was washed with phosphate buffer pH 7.4. It was filled with 3 ml of formulation and suspended in a beaker containing 200 ml of phosphate buffer pH 7.4.  Temperature at about in the shaking incubator was maintained at temperature 37 ^?C with 100 rpm.  Beaker was closed with the aluminium foil to prevent any loss during the experimental run.

3.5.2 Sampling

At predetermined time intervals, 5 ml aliquots were withdrawn from the receptors compartment and were equally replenished with phosphate buffer pH 7.4 and subjected to analysis ¹³. Spectroscopical analysis was carried out immediately after withdrawal of samples with the help of UV – spectrophotometer. The duration of release study was 8 hrs.

1. 6. Formulation of gel (Islam et al., 2004; Das et al, 2007)

As a vehicle for incorporation of niosomes for skin delivery, carbopol gel was made. Carbopol 934 (450 mg) was dispersed in distilled water (60 ml) and allowed to swell overnight. Swelled carbopol was stirred at 800 rpm for 60 min. Mixture was neutralized by dropwise addition of triethanolamine. Mixing was continued until a transparent gel appeared, while the amount of base was adjusted to achieve a gel with pH 5.5^14,15.

3.7 Incorporation of niosomes of Retinol acid and Lincomycin into the carbopol gel

Carbopol 934 (450 mg) was dispersed in distilled water (60 ml) and allowed to swell overnight. The swelled carbopol was stirred at 800 rpm for 60 min. The mixture was neutralized by dropwise addition of triethanolamine. Mixing was continued until a transparent gel appeared, while the amount of base was adjusted to achieve a gel with pH 5.5. Niosomes of Retinol and Lincomycin was dispersed in the carbopol gel with slow agitation.

1. 8. Characterization of gel formulation
      1. Physical examination

The prepared gel formulations were inspected visually for their colour, homogeneity and consistency.

1. 1. 2. pH of gel

pH is a measure of the concentration of hydrogen ions in a solution. Numerically it is the negative logarithm of that concentration expressed in moles per liter.  pH of the prepared gel was measured by a pH meter.

1. 1. 3. Viscosity of gel

Viscosity measurements were carried out at room temperature (25-27 ^?C) using a Brookfield viscometer¹⁵. Sample volume used was 100 ml. Suitable spindle was employed for each treatment, while shear rate was set up at 10 rpm.

1. 1. 4. In vitro skin permeation study (Bachhav et al., 2010)

It is the diffusion of drug across the cellophane membrane into the receptor domain. In vitro skin permeation was conducted on modified Franz diffusion cell. Cumulative amount of drug was assessed by plotting the % cumulative drug permeated against time. Study was conducted for 8 hrs duration. Sampling time was 0, 1, 2, 4, 6 and 8 hrs¹⁶.

4. RESULT (TABLES & GRAPHS) AND DISCUSSION

4.1 Preformulation studies

Preformulation studies were performed for Retinol as well as Lincomycin hydrochloride. Various preformulation parameters such as melting point, IR analysis, solubility, determination of λmax, calibration curve and partition coefficient of drug were determined. Results of these parameters suggested that drug was in pure form.

4.1.1 Physical description

Identification and characterization of Retinol

Identification and characterization of Lincomycin hydrochloride

4.1.2 Melting point

A capillary melting point apparatus was used to determine melting point of Retinol as well as Lincomycin hydrochloride. The observed melting point was 62-64 ^?C and 139-142 ^?C respectively as shown below which is in compliance with standard.

Melting point of Retinol

The melting point of Retinol was found to be 61-65^?C whereas the reported value is 64 ^?C.

Melting point of Lincomycin hydrochloride

The melting point of Lincomycin hydrochloride was found to be 139-144^?C whereas the reported value is 142 ^?C.

4.1.3 Fourier Transform Infra-Red Spectroscopy (FTIR Analysis)

Illustrates FTIR spectra of Retinol. Table shows the frequency of observed bands and its interpretation confirming the purity of sample.

FTIR Interpretation data of Retinol

Illustrates FTIR spectra of Lincomycin hydrochloride. Table shows the frequency of observed bands and its interpretation confirming the purity of sample.

FTIR Interpretation data of Lincomycin hydrochloride

5.1.4 Solubility determination

The quantitative solubility of Retinol and Lincomycin hydrochloride was determined in various solvents at room temperature. Data of solubility in various solvent has been explained in table 14 and 15. These data suggest that Retinol had good solubility in methanol, ethanol and less soluble in water which confirmed its lipophilic nature. In the other hand Lincomycin hydrochloride had good solubility in water and very slightly soluble in ethanol and acetone which confirmed its hydrophilic nature.

Solubility profile of Retinol

Solubility profile of Lincomycin hydrochloride

4.1.5 Determination of λmax

The λmax value for the Retinol as well as Lincomycin hydrochloride was found to be 325 nm and 202 nm respectively. The UV spectrums of both drugs are shown in figure.

UV spectrum of Retinol

UV spectrum of Lincomycin hydrochloride

4.1.6 Calibration curve of Retinol in phosphate buffer (pH 7.4)

Absorbance values of Retinol were taken at 325 nm and plotted with concentrations. Lambert Beer law has obeyed in the concentration range of 10-60 µg/ml with correlation   co- efficient (R²) = 0.997. The standard regression equation was found to be y = 0.010x + 0.137. Calibration data and calibration curves are shown in table respectively.

Calibration data of Retinol in PBS (pH 7.4) at 325 nm

Standard plot of Retinol in PBS (pH 7.4) at 352 nm

4.1.7 Calibration curve of Lincomycin hydrochloride in phosphate buffer    (pH 7.4)

Absorbance values of Lincomycin hydrochloride were taken at 202 nm and plotted with concentrations. Lambert Beer law obeyed in the concentration range of 5-30 µg/ml with correlation co-efficient (R²) = 0.998. The standard regression equation found to be    y = 0.010x + 0.138. Calibration data and calibration curves are shown in table respectively.

Calibration data of Lincomycin hydrochloride in PBS (pH 7.4) at 202 nm

Standard plot of Lincomycin hydrochloride in phosphate buffer (pH 7.4) at 202 nm

5.1.6 Partition coefficient

Concentrations of Retinol as well as Lincomycin hydrochloride in phosphate buffer pH 7.4 were observed at λmax 352 nm and 202 nm respectively. UV absorbance from both the phases i.e. water and n-octanol were taken. Concentration of Retinol and Lincomycin in both phases was estimated and partition coefficient was calculated using the formula, partition coefficient = conc. in organic phase (n- octanol)/ conc. in aqueous phase (water). Data obtained from partition coefficient determination suggest that Retinol is lipophilic in nature and Lincomycin hydrochloride is hydrophilic.

Partition coefficient of Retinol

Partition coefficient of Lincomycin hydrochloride

4.2 Compatibility studies

Compatibility studies were done to check out any physical and chemical interaction between drug and excipients. Samples were observed under all standard conditions for four weeks. The comparison between pure sample and samples with excipients demonstrated no colour change in physical appearance of both drugs. The physical compatibility revealed no change in colour and odour. Further the absence of lumps confirmed that the drug is physically compatible with the excipients. To confirm the results of physical compatibility samples with excipients were subjected thin layer chromatographic analysis on 30^th day. The Rf value of Retinol and Lincomycin hydrochloride was 0.7 and 0.8 respectively. Samples kept for compatibility studies showed same Rf value, confirming the chemical compatibility of both drugs with excipients. Thus, both drugs were fairly compatible with the excipients physically as well as chemically.

Chemical characterization of Retinol - excipients mixtures at different conditions  

(a) 5^?±3 ^?C (b) 25^?±2 ^?C (c) 40^?±2 ^?C

Physical characterization of Retinol - excipients mixtures at different storage conditions on 0^th, 7^th, 15^th, 30^th day

Chol = Cholesterol, PBS = Phosphate Buffer Solution, √ = No physical change

Chemical characterization of Retinol - excipients mixtures at different storage conditions on 30^th day

Chol = Cholesterol, PBS = Phosphate Buffer Solution

√ = No change in Rf value of drug. Rf value in all cases was found to be 0.7 (standard drug Rf is 0.7)

Chemical characterization of Lincomycin hydrochloride - excipients mixture at different storage s (a) 5^?±3 ^?C (b) 25^?±2 ^?C (c) 40^?±2 ^?C

Physical characterization of Lincomycin hydrochloride - excipients mixtures at different storage conditions on 0^th, 7^th, 15^th and 30^th day

Chol = Cholesterol, PBS = Phosphate Buffer Solution, √ = No physical change

Chemical characterization of Lincomycin hydrochloride - excipients mixtures at different storage conditions on 30^th day

Chol = Cholesterol, PBS = Phosphate Buffer Solution

√ = No change in Rf value of drug. Rf value in all cases was found to be 0.8 (standard drug Rf is 0.8)

1. 3. Optimization of surfactant: cholesterol ratio for Retinol

The prepared formulations were evaluated to get an optimized surfactant: cholestrol ratio. Three ratios 1:0.5:1, 1:1:1, 1:2:1 was choosen for the further study. Best ratio was selected on the basis of percentage entrapment efficiency EE (%). 

Entrapment efficiency of different niosomal formulations of Retinol

Graph showing Comparison of various Niosomal formulations of Retinol in term of % Entrapment efficiency

Among all the surfactants, Span 60 demonstrated maximum entrapment efficiency. It was selected as it is solid at room temperature and has highest phase transition temperature (52 °C). Span 60 is a good surfactant as it has a CPP of 0.5-1 and hence forms spherical vesicles.

Entrapment efficiency of different niosomal formulations of Lincomycin hydrochloride

Graph showing Comparison of various Niosomal formulations of Lincomycin hydrochloride in term of % Entrapment efficiency

Niosomal formulations prepared using Tween 60 showed higher entrapment efficiency. Among all the surfactants, entrapment efficiency for niosomes prepared using Tweens was superior to those prepared using Spans. This can be explained by the fact that large hydrophilic head Tweens is capable of solubilizing greater amount of Lincomycin hydrochloride which is extremely hydrophilic. Furthermore with increase in concentration of surfactants (Tweens, Spans), entrapment efficiency was found to be increased.

4.4 Evaluation parameters of niosomes

4.4.1 Particle size measurement and zeta potential

Particle size of the optimized formulations of Retinol and Lincomycin hydrochloride was found to be 507.8 nm and 512.8 nm with polydispersity index of 0.826 as well as 0.611. Zeta potential of the optimized formulations was found to be -6.28 mV and -12.8 mV. Particle size of the particulate system generally affects its penetration into skin

Zeta potential distribution curve for Retinol

Zeta potential distribution curve for Lincomycin hydrochloride

4.4.2 Optical microscopy

Photomicrographs of niosomes of Retinol and Lincomycin hydrochloride were obtained by optical microscope. The results revealed the presence of uniform, spherical single layered vesicles (Unilamellar).

4.4.3 In vitro drug release study

Comparative % age cumulative drug release profile of drug solution and niosomal solution is shown in Tables. Drug release of the niosomes loaded with Retinol as well as Lincomycin hydrochloride was carried out in phosphate buffer at pH 7.4 for 8 hrs. As it is clear from the Fig. 28-29, the release rate of niosomal formulation was slower than observed with drug solution.

In vitro comparison of drug release for niosomal solution and plain drug solution (Retinol)

Comparative in vitro drug release profile of niosomal solution and plain drug solution (Retinol)

In vitro comparison of drug release for niosomal solution and plain drug solution (Lincomycin hydrochloride)

Comparative in vitro drug release profile of niosomal solution and plain drug solution (Lincomycin hydrochloride)

4.5 Results for gel formulation

4.5.1 Physical examination

The prepared gel formulation was observed visually and was found to be transparent white.

4.5.2 pH of gel

The pH of the gel formulation was found to be 6.1 Thus, the formulation is dermatologically compatible.

4.5.3 Viscosity

Viscosity of prepared gel was found to be 0.5284 (Pa/s). It was determined with the help of Brookfield DV-1 viscometer. It affects the spreadibility and adherence of transdermal formulations to the skin surface.

4.5.4 Permeation study

Cumulative amount of drug loaded in niosomal gel was assessed by plotting the % cumulative drug permeated against time. It showed better skin permeation in 8 hrs.

In vitro permeation studies

1. 1. 5. CONCLUSION