Formulation And Evaluation of Nanosponges Loaded Tretinion Gel for The Treatment of Acne

SKIN:

The biggest organ in the body, skin accounts for 10% of body weight and connects the body to the outside world. It act as a obstacle between body& the outside world to protect both. The surface area of skin 1.5 to 2.0 sq. meters whereas the thickness lies between 0.5 – 3mm. Skin conditions are prevalent across different cultures, ages and socioeconomic statuses, making one of the most widespread medical issues worldwide. The epidermis, dermis, and hypodermis are the three layers of skin. Apocrine glands, sweat ducts, and hair follicles are further connected appendages of the skin. Numerous compounds are intentionally or unintentionally applied to the skin, either for good or bad results. Topical pharmaceuticals have been used historically, and information from the 1940s showed that hormone and anti-infective topical therapies were popular at the time.

Main areas of use of dermal preparations are:

• Local effects
• To transport drug to the skin (like- Nicotine patches for skin)
• To make effects of the preparation on the surface of skin to target deeper tissues (Sunscreens, Cosmetics and Anti-infectives).
• To neglect the problems associated with the stomach emptying, pH, deactivation of enzymes, and first pass metabolism.
• To avoid the first pass metabolic effects of skin.
• Excretion: Water, salts, and trace amounts of nitrogenous waste are all eliminated via the skin.
• Sensation: The skin is responsible for sensing different types of stimuli such as: touch, temperature.
• Immune defense: Skin serves as a barrier against infections and contains immune cells that help defend against pathogens.

Fig. 1.1. Gross Skin Structure

An excellent example of this is how the skin's barrier properties prevent the entry of a chemical compound, metabolism bypasses stratum corneum &attention is then drawn to the smash up this entry causes (inflammatory mediators released in the epidermis) and the removal of this chemical compound with the supply of dermal blood &distribution in the body organs responsible for its purging either by meta- or endo-metabolism. Skin has mainly 3 layers:

Epidermis: The creation of the echelon corneum is one of the epidermis many tasks, which makes it the top layer and one of its most vital ones. Its job is to shield the body from the elements and create a watertight barrier.

Fig. 1.2 Structure of Epidermis

Stratum Spinosum:

This layer is finished up of several layers of living cells coupled by desmosomes, which are protein-based structures that aid in holding the cells together. Epidermis' deepest layer and are in charge of creating new skin cells, are located in the stratum basale. Melanocytes, which are found in this layer as well, are responsible for producing the colour. The heterogeneous epidermis' uppermost layer, the stratum corneum, has a thickness that ranges from 10 to 30 micrometres. It consists of 15–25 cornified, layered, flattened, hexagonal cells that are enclosed in an intercellular lipid mortar. The thickness of the stratum corneum varies, nevertheless.

Dermis

The dermis plays a critical function in the regulation of temperature, pressure, and pain. It also aids in nourishment, immunity, and many other supportive systems for the epidermis thin papillary layer that is next to the epidermis. According to some experts, the dermis' primary structural element is a rough reticular layer. Its thickness ranges from 0.1 to 0.5 cm, & its composition is 70% collage nous fibres, which offer prop up and cushion, and 20% flexible connective tissue, which is responsible for giving elasticity in a mucopolysaccharide semi-gel matrix.

Sub-cutaneous tissue:

The hypodermis serves as an energy storage area as well as a heat and shock absorber. The dermis and hypodermis are connected by interfacial collagen and elastin fibres, which is a complex of lobulated fat cells. The hypodermis' other main cells are fibroblasts and macrophages, in addition to fat cells (which may make up 50% of the body's fat).

Skin Appendages

Skin appendage come in four different varieties: Hair follicles and associated oil glands Endocrine sweat glands. Sweat glands called apocorins and Nails Targeted drug shipping systems of this kind have some of fundamental benefits. As the drug is discharged on the tumor website online in preference to circulating broadly through the body, it must be more useful for a given dosage. They also must have fewer harmful adverse results since lower amounts of the active drug come into touch with strong tissue. Another gain is that the nano-sponge debris are dissolved in water. Encapsulating the anticancer drug in nano-sponge grant using hydrophobic capsules that don't dissolve in water. Newly, these pills ought to be joint with adjuvant reagents, which probably can lower the efficacy of the drug or cause damaging effects (1).

Nanosponges are highly effective drug delivery systems that release therapeutic agents directly at the targeted site, reducing the need for widespread circulation throughout the body. This targeted approach increases efficacy at lower doses and minimizes side effects, as less of the drug interacts with healthy tissues. Unlike many nanoparticle systems that release their payloads rapidly and unpredictably—a phenomenon known as the "burst effect"—nanosponges enable controlled, sustained drug release, making dosage levels easier to manage.

These structures feature numerous functional groups that simplify the attachment of drug molecules. Their surface chemistry can be easily modified to adjust key properties such as hydrophobicity, morphology, particle size, and functionality. This adaptability allows nanosponges to be optimized for specific therapeutic needs. In contrast to traditional polymer nanoparticles, which often require complex and difficult post-synthesis modifications, nanosponges can be functionalized with relative ease.

Features of nanosponges

A valuable character of those sponges is their aqueous solubility; this confesses the use of those systems correctly for tablets with poor solubility. The Nanosponges are capable to circulate each hydrophilic and lipophilic drug. Elimination of organic impurities in water, as nano-companies for biomedical utilization. This era gives entrapment of components and decreased side consequences, increased elegance, improved stability, and more suitable method flexibility. Nanosponges are non-mutagenic, and non-demanding, reliable and non-allergic. Extended release non-stop release up to 12hg rantin corporation of immiscible liquid better cloth processing-liquid can be transformed to powders. They may be restoration in a sub microns spherical particle. They can be gathering in a extensive range of dimensions, from 1micron to10microns.The cavities of the structure have at unable polarity.

Advantages of nano-sponge drug-delivery system

• Allows fusion which improve smatter processing-liquid can be changed to powders.
• These formulation sares table over broad range of pH and temperature.
• These are self-sterilizing as their average pore size is 0.25µm where bacteria can not enter.
• These are free flowing greatly    compatible with wide diversity of ingredient and charge valuable.
• Particles can be formed in error bigger by changing the proportion of cross-linked polymer.
• Simple scale-up for commercial manufacturing.
• Biodegradable.

Applications of Nanosponges

Solubility enhancement:

Nanosponges can significantly enhance the wetting properties and solubility solubility. molecularly dispersed within the nanosponge matrix, allowing them to be released in molecular form and bypass the traditional dissolution step. As a result, the apparent solubility of the drug increases. Enhancing solubility and dissolution rate is a key strategy for addressing formulation and bioavailability challenges, and nanosponges provide a promising solution in this regard.

Nanosponges for drug delivery:

Nanosponges are solid in nature and can be formulated for various routes of administration, including oral, parenteral, topical, and inhalation delivery. For oral use, nanosponge-drug complexes can be incorporated into a mixture of excipients, diluents, lubricants, and anti-caking agents suitable for the preparation of tablets or capsules. In parenteral formulations, the nanosponge complexes can be simply suspended or dissolved in sterile water, saline, or other appropriate aqueous solutions for injection. For topical management they may be efficiently integrated into topical hydrogel.

Topical sellers:

The nanosponge delivery-system is an advanced technology designed for the controlled release of topical agents, enabling prolonged drug retention and sustained release on the skin's surface. This approach is particularly effective for formulating drugs such as local anesthetics, antifungals, and antibiotics, which benefit from extended action at the site of application and minimal systemic absorption. Rashes or more important element effects can occur even as energetic components penetrate the pores and skin. In assessment, this technology lets in an first rate and sustained charge of release, decreasing irritation even as maintaining overall performance. Nanosponges have shown great potential as carriers for biocatalysts and for the delivery and controlled release of enzymes, proteins, vaccines, and antibodies. Various delivery systems, including nanoparticles, microparticles, liposomes, and hydrogels, have been explored for transporting proteins and enzymes. These systems can enhance the stability of biological molecules, protect them from degradation, and modulate their pharmacokinetic profiles.

Nanosponges as a carrier for shipping of gases:

Gases are critical in medical applications, serving both diagnostic and therapeutic purposes. A deficiency in oxygen supply, known as hypoxia, is associated with a wide range of conditions, including inflammation and cancer. However, delivering oxygen in a controlled and effective manner can be challenging in clinical settings. To address this, Cavalli and colleagues developed nanosponge-based formulations designed for topical oxygen delivery. These systems are capable of storing oxygen and releasing it gradually over time, offering a sustained and controlled method for oxygen administration. (9).

Nanosponges as protecting agent in opposition to picture degradation:

Sapino et al. proposed the use of gamma-oryzanol—a mixture of ferulic acid esters known for its antioxidant-properties and commonly used to stabilize food and pharmaceutical raw materials—as a sunscreen agent in cosmetic formulations. However, its broader application is limited due to its high susceptibility to instability and degradation upon exposure to light. To overcome these challenges, gamma-oryzanol was encapsulated within nanosponges, which significantly improved its photostability. Using these gamma-oryzanol-loaded nanosponges, both a gel and an oil-in-water (O/W) emulsion were successfully formulated for topical application.(9).

Removal of Organic Pollutants from Water:

Beta-cyclodextrin-based nanosponges are entirely insoluble in water and possess the ability to encapsulate organic contaminants, making them effective for water purification applications. These nanosponges can be integrated into ceramic porous filters, creating hybrid organic/inorganic filtration systems. Such hybrid filter modules have been tested and demonstrated high efficiency in removing various pollutants from water. Notably, they have shown over 95%. In addition, they are effective in eliminating other contaminants, including trihalomethanes (THMs), monoaromatic hydrocarbons (BTX), and pesticides such as simazine.

Topical Gel

Topical gels are semi-solid formulations intended for application on the skin or mucous membranes. They are characterized by their unique consistency, which is achieved by dispersing active pharmaceutical ingredients (APIs) in a suitable gelling agent such as carbomers, cellulose derivatives, or natural gums. These systems are primarily aqueous but may contain alcohol or other co-solvents to enhance solubility and drug permeation. Topical formulations like creams or ointments. They are non-greasy, easily spreadable, and quickly absorbed without leaving a sticky residue, making them highly acceptable to patients. Additionally, their high water content can have a soothing effect on inflamed or irritated skin, which is particularly important in dermatological therapies. From a pharmaceutical perspective, gels allow for precise drug dosing and rapid release of active ingredients at the site of application. This is especially important in treating localized skin disorders where systemic exposure is unnecessary or potentially harmful. However, formulating a stable and effective topical gel presents its own set of challenges, especially for labile or poorly soluble drugs like tretinoin. Due to their favorable properties and ease of application, gels have become one of the most popular vehicles for dermatological drugs, including anti-acne agents, antifungals, corticosteroids, and retinoids. For tretinoin, gels can enhance skin penetration but are also more likely to cause irritation compared to creams or lotions—highlighting the importance of optimizing gel composition and release profiles to improve both efficacy and tolerability.

Challenges in Topical Delivery of Tretinoin

The primary challenges in the topical administration of tretinoin include:

• Skin Penetration: The stratum corneum acts as a formidable barrier, limiting the penetration of many therapeutic agents, including tretinoin.
• Photodegradation: Tretinoin is susceptible to degradation upon exposure to light, reducing its efficacy.
• Local Irritation: Common side effects such as erythema, peeling, and dryness can lead to patient non-compliance.

MATERALS AND METHODS

Table 2.1: Composition of gel formulation

PRE-FORMULATION STUDIES

Organoleptic Properties

Organoleptic Houses of Tretinoin will achieved by human sensory organs. The organoleptic research of Tretinoin like popular appearance like color, scent, us of a, and lots of others. Will finished.

Solubility study

The qualitative solubility of tretinoin in various solvents will be determined following the guidelines outlined in the USP NF, 2007. Approximately 1 mg of tretinoin will be weighed and transferred into a 10 mL test tube, where it will be dissolved in 1 mL of each solvent, including methanol, ethanol, DMSO, chloroform, and water.

Melting Point

The melting point will be determined using the open capillary method with Thiele’s tube. A small amount of tretinoin will be placed into a thin-walled capillary tube, 10-15 mm in length and approximately 1 mm in internal diameter, with one end sealed. Liquid paraffin oil will be added to the Thiele's tube, which is then placed over a flame. The capillary tube containing the sample will be suspended in the Thiele’s tube, and the sample will be heated slowly. A thermometer will be used to monitor the temperature, and the temperature at which the sample begins to melt will be recorded as the melting point. (16).

Partition coefficients

The partition coefficient of drug Tretinoin will examine in, n-Octanol: water system. It will determined by taking 5mg of Tretinoin (drug) in separating funnel containing, 20ml of n-Octanol and 20 ml water. The separating funnel will shake for 2 hours in a wrist action shaker for equilibrium. Two phases were separated and the amount of drug in aqueous phase will analyzed spectro photo metrically at 294.0 nm. The partition coefficient of drug formula-(16).

Determination of Lambda-max and calibration curve:

Preparation of standard stock solution:

About 5mg of Tretinoin will weighed and transferred into 5ml volumetric flask. The quantity will made as much as 5ml using methanol to achieve an answer that has a concentration a thousand µg/ml. 1ml of this inventory answer will taken after which diluted up to ten ml the use of methanol to gain a solution that has a awareness 100 µg/ml that's standard inventory solution.

Lambda max

From the above stock solution, 2 mL of the sample will be transferred into a 10 mL volumetric flask, and the volume will be adjusted to the mark with methanol to prepare a concentration of 20 µg/mL. The sample will be analyzed using a double-beam UV-VIS spectrophotometer (Shimadzu - 1700) in the wavelength range of 200-400 nm, with methanol used as the blank. The maximum absorbance (λmax) of the sample will be recorded.

Linearity (Calibration curve)

Aliquots of 5, 10, 15, 20, 25, 30, and 35 µg/mL will be prepared from a 100 µg/mL stock solution of tretinoin. The required volumes of the stock solution will be transferred accurately into a series of 5 mL volumetric flasks, and the volume will be adjusted to the mark with methanol. The absorbance of these solutions will be measured at 294.0 nm against a methanol blank. A calibration curve will be constructed by plotting absorbance versus the concentration of the drug. The calibration curve will consist of seven data points within the concentration range of 5-35 µg/mL of tretinoin.

Fourier-transmission Infra-Red Spectroscopy

The FT-IR spectrum of tretinoin was recorded in the range of 4000 to 400 cm?¹ using the KBr pellet method with an FT-IR spectrophotometer. To prepare the KBr disc, 1 mg of tretinoin was mixed with 100 mg of spectroscopic-grade KBr, which was dried using an IR lamp. The mixture of KBr and the drug was then subjected to hydraulic pressure to form a disc. This disc was placed in the FT-IR chamber, and the infrared spectrum was recorded within the range of 4000 to 400 cm?¹. (16).

Formulation of β-cyclodextrin Nano sponges

Beta-cyclodextrin Nano sponges will be prepared using the 'hot melt method'. Different ratios of diphenyl carbonate (DPC, the cross-linker) and beta-cyclodextrin (β-CD, the polymer) will be selected for the preparation, as indicated in Table 1. The anhydrous β-CD polymer and DPC cross-linker will be finely homogenized and gradually heated to 90-100 ºC with continuous magnetic stirring for 6 hours. The reaction mixture (β-CD and DPC) will be allowed to react for 6 hours to ensure the completion of the crosslinking reaction, resulting in the formation of nanosponges. Afterward, the reaction mixture will be cooled to ambient temperature. The resulting solid will be washed multiple times with double-distilled water to remove any unreacted β-CD. Finally, the placebo nanosponges will be dried at 40 ºC and stored in a desiccator until further use. (19).

Loading of Tretinoin in Nano sponges

Tretinoin will be loaded into the prepared nanosponges via lyophilization. One gram of placebo nanosponges (NS) will be dispersed in 50 mL of double-distilled water with the help of a magnetic stirrer. Approximately 50 mg of tretinoin will be added to this dispersion. The resulting mixture will be sonicated for 10 minutes and then stirred for 24 hours. Afterward, the suspension will be centrifuged at 2000 rpm for 10 minutes to separate any un-entrapped tretinoin, which will form a residue beneath the colloidal supernatant. The supernatant will be lyophilized at -81 ºC and 0.0010 mbar pressure. The dried tretinoin-loaded nanosponges powder will be stored for future use. (19).

Evaluation parameters of Tretinoin loaded nanosponge

Zeta-Potential

The zeta potential will be measured to determine the movement rate of particles in an electric field and their surface charge. In this study, the nanosponges will be diluted 10 times with distilled water and analyzed using a Zetasizer Malvern instrument. Prior to measurement, all samples will be sonicated for 5-10 minutes to ensure uniform dispersion.

Particle Size

The particle size of the nanosponges will be measured using a Malvern ZetaSizer (Malvern Instruments). The dispersions will be diluted with Millipore-filtered water to an appropriate scattering intensity at 25°C. The sample will then be placed into a disposable sizing cuvette for analysis.

Entrapment Efficiency

To determine the entrapment efficiency, 10 mg of the nanosponges will be weighed and dispersed in 5 mL of methanol in a volumetric flask. The mixture will be vortexed for 1 minute, and the volume will be adjusted to 10 mL. The solution will be filtered and further analysis.

Loading Efficiency = (Actual drug content in nanosponges / Theoretical drug content) × 100

Formulation of Nanosponges-Loaded Gel.

A permeation enhancer, propylene glycol, will be added, and the final dispersion will be stirred until a smooth gel is formed, free of lump

Characterization

Physical-appearance

The prepared Gel formulation will evaluated for appearance, Color, Odor, and homogeneity by visual observation (27).

pH

pH of the formulation will determined by using Digital pH meter (EI). The meter will allowed to stabilize as necessary and properly calibrated, begin by rinsing the probe with de-ionized or distilled water and blotting the probe dry with lint-free tissue paper. Immerse the sensing tip of the probe in the sample and record the pH reading and Rinse the probe, blot dry and repeat step 2 on a fresh portion of sample. The two readings should agree to within the accuracy limits of the meter. The samples will analyzed in triplicate. If slight deviations in pH were noted, it will adjusted to skin pH using drop wise addition of tri-ethanolamine solution.

Spreadability

The time taken for the gel to travel the distance from the place of its position will noted down. Spreadability will determined by the following formula

S= M*L/T

Where, S-Spreadability, g.cm/s M-Weight put on the upper glass L-Length of glass slide T-Time for spreading gel in sec.

In-vitro drug release 

The study of the nanosponges-loaded gel formulations will be conducted using the dialysis bag diffusion method. The gel containing the nanosponges will be placed inside a dialysis bag, which is then submerged in a beaker containing 100 mL of pH 7.4 phosphate buffer. The beaker will be positioned on a magnetic stirrer, and the temperature will be maintained at 37 ± 2°C throughout the experiment. The stirring speed will be set at 100 rpm. At specified time intervals, 2 mL samples will be withdrawn and replaced with an equal volume of fresh pH 7.4 phosphate buffer. After appropriate dilution, the samples will be analyzed using a UV–Visible spectrophotometer at 294.0 nm. The zero-order kinetic model (Equation 2) is used to describe systems where the drug release rate is independent of its concentration. The first-order model (Equation 3) is applicable to systems where the release rate depends on the concentration of the drug. Higuchi’s model describes the release from an insoluble matrix, assuming it follows a time-dependent process based on Fickian diffusion. The in-vitro release profiles obtained for all formulations will be plotted and analyzed using the following models:

Zero order kinetics

Zero order release would be predicted by the following equation:

Where

At= Drug release at time‘t’

A0= Initial drug concentration.

K0= Zero-order rate constant (hr-1)

First-order kinetics

LogC=logC0–Kt/2.303-------- Eq. (3)

First-order release might be expected by the subsequent equation:

Where,

C = Amount of drug remained at time ‘t’

C0 = Initial amount of drug.

K = First-order rate constant (hr?¹).

When the data is plotted as the log of cumulative percentage of drug remaining versus time, it results in a straight line, indicating that the drug release follows first-order kinetics. The rate constant 'K1' can be determined by multiplying 2.303 with the slope value obtained from the plot.

RESULT

Pre formulation study:

The organoleptic houses of tretinoin display faded yellow to yellow crystal.

Solubility profile:

Tretinoin turned into practically insoluble in water, sparingly soluble in chloroform, and soluble in alcohol, glycerin, isopropyl alcohol and polyethylene glycol four hundred. Data became shown in Table 3.1.

Table 3.1 : Solubility profile of Isotretinoin

Study of drug-excipient interaction:

Drug excipient interaction became finished in exceptional condition (room temperature, 40°C/seventy fivepercentRH & 30°C/60%RH) for 4 week and shown in Table 6.2  The rejection and choice of high-quality components is based on the texture and coloration adjustments. The result suggests that no change in coloration turned into visible after four weeks.

Table 3.2: Observations of drug – excipient interaction

Infrared Spectroscopy (FT-IR study):

The FTIR spectra of pure drug, excipient and polymer were recorded in four hundred to 4000 cm-1 on IR Bruker Alpha software program. No interference peaks are discovered with drug (Figure 6.1) and different excipient. The distinctive peaks acquired are summarized in Table 6.3.

Table 3.3: FT-IR peaks of tretinoin with other excipient

(a)

(b)

Figure 3.1: Infrared spectrum of (a) tretinoin and (b) mixture of all excipient with tretinoin.

UV Analysis:

The calibration curve of Isotretinoin turned into analyzed with the aid of extremely-violet spectroscopy and analyzing became shown in Table 6.Four and popular curve of Isotretinoin by UV changed.

Table 3.4: Standard curve for tretinoin

Figure 3.2: Standard curve of tretinoin by UV spectrophotometer

DSC (Differential Scanning colorimetry):

Differential Thermal Gravimetric evaluation and thermal gravimetric analysis changed into also completed for the drug and proven in Figure 3.3.

Figure 3.3: Infrared spectrum of tretinoin

Tretinoin Normal Gel-Formulation (NGF1to NGF9)

Drug content material uniformity, UV evaluation, In-vitro drug launch, drug content material, diffusion have a look at and In- vivo pores and skin infection study.

Physicochemical evaluation

Solubility

The gel demonstrated solubility in various organic solvents but was found to be insoluble in distilled water. During the solubility assessment, it fully dissolved in toluene, PEG-400, glycerin, petroleum ether, propylene glycol, and acetone. Partial solubility was observed in methanol and ethanol, while negligible dissolution occurred in distilled water.

Stickiness

The results indicated that the formulated tretinoin gel was non-sticky upon application. It spread easily on the skin and was compared favorably with the commercially available formulations.pH of the tretinoin-gel

3.5 table: observation of pH

Viscosity

The viscosity of drug formulations was measured using a Brookfield analog viscometer, with the results presented in Table 3.6

TABLE: 3.6 VISCOSITY OF FORMULATION

Spreadability

The spreadability of the formulations was found to range between 15.508 and 48.623 cm/second. Batch NGF4 and NGF5 exhibited lower viscosity compared to the other batches, resulting in better spreadability, which was lower than the marketed product (Sortet gel: 52.416 g/cm³). Among all the formulations, NGF5 demonstrated the highest spreadability. The average spreadability values for all formulations are presented in Table 3.7

Table 3.7  average spreadability of Formulation

Drug Content uniformity (Assay)

The drug content of the gel formulations was consistent across the various batches, ranging from 89.93% to 96.70%. Notably, the formulation batches NGF4, NGF5, NGF6, and NGF7 demonstrated drug content above 95%. The content uniformity of the gel formulations is presented in Table 3.8. Among these, formulation NGF5 exhibited a drug content of 99.2%.

TABLE 3.8 Drug Content uniformity

In-vitro drug release

The study of the nanosponges-loaded gel formulations will be conducted using the dialysis bag diffusion method. The gel containing the nanosponges will be placed inside a dialysis bag, which is then submerged in a beaker containing 100 mL of pH 7.4 phosphate buffer. The beaker will be positioned on a magnetic stirrer, and the temperature will be maintained at 37 ± 2°C throughout the experiment. The stirring speed will be set at 100 rpm.

TABLE 3.9 INVITRO DRUG RELEASING

Figure 3.4: Drug release profile comparison with marketed product