Dacarbazine was obtained from Mumbai-based Neon Laboratories Limited. BASF Chemicals, Bengaluru, India, supplied Kolliphor^® P188. Sigma-Aldrich, Bengaluru, India, supplied glycerol monooleate, phosphatidylcholine, ethyl alcohol, and polysarcosine. Sisco Research Laboratories Pvt. Ltd., Mumbai, supplied the gellan gum, chitosan, and phosphate buffer tablets. Sigma-Aldrich, Bengaluru, India, supplied the Gold (III) chloride trihydrate (HAuCl4.3H2O). Rest all or most of the chemicals and reagents of specific analytical grades that have been used.

The lyophilized Dacarbazine encapsulated shell-enriched solid lipid nanoparticles (40mg) were dissolved in 20mL of 75% v/v methanol, and the temperature was maintained up to 65°C in a water bath for 45min. The lipid started leached out when the temperature was reduced to 20-25°C. Furthermore, the obtained mixture was centrifuged at 6000 rpm. The obtained supernatant was diluted in distilled water and spectroscopically by LAMBDA XLS+Spectrometer (PerkinElmer Fremont, CA, USA) at 328 nm. As per the method described in Mihaela chişa, et al., 2016 (35), entrapment efficiency was performed. The entrapment efficacy of the prepared formulation was calculated by following equations:

Delsa Nano C instruments (Beckman Coulter, USA) measure the particle size, polydispersity index (PDI) and zeta potential of the prepared DTIC-SLNs formulations. The emulsion of the nanoparticles was diluted with 0.57 mL of double distilled water and vortexed for 4 minutes to prevent bubbling. The particle size, PDI were measured using Dynamic Light Scattering (DLS) and Zeta potential was measured by Electrophoretic Light Scattering (ELS) at 25°C.

TEM electron microscopy (TEM) was used to determine the morphology of the DTIC-SLNs formulations and obtain high-quality images. The TEM instrument used was a Hitachi 7500 from Japan. Prior to TEM analysis, the nanoparticles were coated with carbon and placed on a stained copper grid coated with phosphotungstic acid (1%). The images obtained from TEM studies were analyzed using digital micrograph-generated software.

Using a modified Franz cell system (36), the permeation studies of DTIC-SLNs formulations was investigated. In the upper donner compartment, 0.2µm cellulose nitrate membrane were placed, which eventually sperate the donor compartment and receptor compartment. The 10mg DTIC-SLNs were suspended into a 2mL phosphate buffer solution and placed into the donor compartment. The receptor compartment was filed with 20mL of pH 7.4 phosphate buffer solution. While stirring at 100rpm, a 0.5mL sample was extracted from the receptor compartment at a specific time interval and filtered. The filtrated was measured spectroscopically (PerkinElmer Fremont, CA, USA) at 328 nm. The cumulative amount of the drug permeated per unit area of the nitrate membrane after 24h (Q24 in μg/cm2), the permeation initiation time (lag time in min), and permeability coefficient (Kp) were calculated. The effects of gel base on permeability can be measured by incorporating 25mg of DTIC-SLNs-9 formulation into multiple cationic gel bases, i.e., Gellan gum (0.01%w/v), Chitosan (0.5% w/v), Polysarcosine (2.5% w/v), respectively. Prior to use, the prepared gels were left to equilibrate at 0-4°C for 24h.

Stability studies were conducted as per International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH). Pharmaceutical development: Q8(R2) current step 4 version. Geneva: ICH; 2009.guidelines (37). Measuring UV-VIS spectroscopic encapsulation efficacy (PerkinElmer Fremont, CA, USA) at 328 nm and investigating zeta-potential and particle size of optimized DTIC-SLNs-8 utilizing Delsa Nano C instruments (Beckman Coulter, USA) for 9-months, the stability studies were performed.

The 10% formalin-fixed tumor tissues were embedded in paraffin and sectioned at 4 µm. The slides were deparaffinized and rehydrated with ethanol. Next, the slides were incubated with anti-Ki-67 primary antibody. Followed by HP/streptavidin conjugated secondary antibody, DAB substrate were added to the slides and counterstained with hematoxylin. The slides were then observed under optical microscope. To calculate proliferative indices, 15 randomly selected microscopic (40× objective) fields in each group were calculated by the total number of cells divided by the number of Ki-67- positive cells.

Statistical Analysis All data are expressed ad mean ± standard deviation (SD). Significant differences were assessed using Student’s t-test and p value < 0.05 was considered as statistically significant.

The collected data were statistically evaluated using a t-test for unpaired data; p<0.05 was considered significant.

Riddick, 1968 first defined the theshold zeta potential value of agglomeration within the dispersion, which is in between -20 mV to -11mV. As per Sobia Razzaq et al. (2021), paclitaxel multi-functional papain anchored polymeric amphiphilic micelles were papered. Which had negative zeta potential (−29 ± 3.85mV) with higher encapsulation efficacy (80 ± 3.29%) (40). Putthiporn Khongkaew et al. (2021) combined propolis wax with surfactants such as Brij 721, Cremophor WO 7, Myrj 52, Kolliphor^® P188, and Tween 80 to develop Solid Lipid Nanoparticles (SLN). Brij 721 and Myri 52, when used at a 20% concentration, can be used to make SLN and have good vitamin E preservation properties (size: 451.2 nm and 416.8 nm, zeta potential: - 24.0mV and - 32.7mV, respectively; percent EE: 92.32% and 92.00%) (41).

In the current experiment, except DTIC-SLNs-8 to DTIC-SLNs-11, rest all the formulations zeta potential are relatively low and within the Riddick theshold (-19.56 ± 2.82mV to -12.45 ± 2.78 mV). Due to the shielding effects of nanoparticles owing to the hydrophilic corona of Kolliphor^® P188, the zeta potential values of nanoparticles are inadequate to produce electrostatic stabilization. However, from the transmission electron microscopy, it is clearly indicating that, Kolliphor^® P188 offers additional steric and electrostatic stabilization within formulae. The presence of Kolliphor^® P188 increases steric stabilization within the surface of the nanoparticles, even though the particles have an inadequate charge to generate electrostatic stabilization. Particle size analysis by though Dynamic Light Scattering (DLS) system indicating that ( Table 2 ), almost all the formulation had smaller particle size ranging from 146 ± 4.71 nm to 415 ± 10.89 nm and PDI from 0.17 ± 0.013 to 0.28 ± 0.012. However, formulation DTIC-SLNs-10 & DTIC-SLNs-11 showing aggravated zeta potential, particle size and polydispersity index, which is due to the absence of Kolliphor^® P188 and phosphatidylcholine within one of each formulation. In comparison to the average mean diameter and surface area of the collected morphological images from TEM analysis ( Figures 2A–D ), the average mean diameter obtained by the Dynamic Light Scattering (DLS) process was slightly higher. Reversed micelles are water droplets with a diameter of 1-10nm that form in the presence of a suitable surfactant. The surfactant molecules exposed the organic phase by rearranging polar parts to the inner side and non-polar regions. The nature of the surfactant used during the preparation of reversed micelles, according to Mehta, Ganguli et al. (2021), will influence the shape of the formulation. Hydrophobic alkyl chains and cationic, anionic, and non-ionic hydrophilic head groups were used to create reversed micelles in this experiment. Spherical shell micelles were studied using Small Angle X-ray Scattering (SAXS) (42).

In the current experiment, due to the steric hindrance, the aggregation tendency of the nanoparticles was less prominent. The use of Small Angle X-ray Scattering (SAXS) to examine the morphological behaviour of reversed micelles was not considered in this study. However, using transmission electron microscopes (TEM) the micelle formation can be theorized and examined. From the TEM image ( Figure 2D ) It was assumed that nanoparticles had developed two distinguished layers, with the outer hydrophilic layer representing the shells and the inner hydrophilic layer representing the core, composed of loaded Dacarbazine (DTIC). Dacarbazine (DTIC) is believed to be present in these layers or shells (43). The presence of Kolliphor^® P188 and phosphatidylcholine might helps in to formulate micelles surrounding of core comprising of loaded Dacarbazine (DTIC). Therefore, an adequate number of DTIC loaded overturned micelles might be seen near the core-shell region; the TEM image also confirmed the formation of such micelle’s formation ( Figures 2B , 3 ).

Reverse micelles are made in a unique ways, according to R M de Souza et al. (2020) ELBA (44), coarse-grained force can be used to check the self-assembly of 2-dipalmitoyl-sn-glycero-3-phosphocholine/1, 2-dihexaoyl-sn-glycero-3 phosphocholine(DPPC/DHPC)bicelles. The results of the atomic experiment suggest that disclike shape reverse micelles can be prepared with a higher coarse-grained force and a correct aggregation number while using a higher coarse-grained force.

However, some speculated models are postulated, which support drug distribution theory, i.e., (a)the drug enriched within the core-shell, (b). The partially aqueous soluble DTIC dissolved in ethyl alcohol, organic solvent, and subsequently added into warm dextrose 5% water to form reverse micelles. Therefore, it is essential to have good compatibility between hydrophobic surfactant phosphatidylcholine and hydrophilic surfactant called Kolliphor^® P188. To form a good lipid matrix, both hydrophobic and hydrophilic surfactants ratio optimization is required. Since phosphatidylcholine is strongly lipophilic, it can attempt to dissolve and encapsulate DTIC. The hydrophilic nature of Kolliphor^® P188, on the other hand, can shape fabric-like projection structures around the DTIC-SLNs, preventing micelles from dissolving in aqueous environments. The fraction of Kolliphor^® P188 that remains in the aqueous solvent during the SLNs preparation process, in principle, determines the encapsulation efficiency of hydrophobic DTIC. The entrapment efficacy (%) and particle size findings of DTIC-SLNs-10 and DTIC-SLNs-11 indicate that in the absence of phosphatidylcholine and Kolliphor^® P188, the solid lipid nanoparticles’ entrapment efficacy (%) decreases and particle size increases. This clearly indicates phosphatidylcholine and Kolliphor^® P188 can entrap micelles within the shell region, thus preventing the escape of micelles.

Moreover, 5.5, 7,5 and 10 mg for Phosphatidylcholine and as 1.1, 1.2 and 1.4% for Kolliphor^® P188 ratio had a great influence on entrapment efficacy (%), particle size (nm) and zeta potential (mV). In DTIC-SLNs-1, as the lower ratio of Phosphatidylcholine and Kolliphor^® P188 (5.5:1.0) resultant in moderately higher particle size (415 ± 10.89nm), low entrapment efficacy (51.56 ± 1.78%) and unstable zeta potential (-12.45 ± 2.78mV); this phenomenon might be due to the uneven coating of glycerol monooleate over SLN in the presence of lower Phosphatidylcholine and Kolliphor^® P188 ratio. However, in formulation DTIC-SLNs-9, where the higher ratio of Phosphatidylcholine and Kolliphor^® P188 (10:1.4) incorporated, resulted in higher entrapment efficacy (87.45 ± 4.78%), moderately low particle size (198 ± 4.35nm) and highly stable zeta potential (-25.56 ± 2.78mV); this might be due to the presence of steric repulsion between the particles and appropriate coating of glycerol monooleate while formulating SLN.

However, TEM images of DTIC-SLNs-10 and DTIC-SLNs-11 showing larger particle size with the absence of micellar formation. To understand the morphological behaviour of the DTIC-SLNs-10 and DTIC-SLNs-11, 3D surface images were plotted using ImageJ software ( Figures 4B, D ). It is evident from Figures 4A, C that SLNs without phosphatidylcholine and Kolliphor^® P188 may have agglomerated mushooms effects due to their larger particle size.

It was also understood that in the absence of one of both, phosphatidylcholine and Kolliphor^®, SLNs could not produce micelles within and hance, entrapment efficacy (%) became limited. Moreover, the DTIC-SLNs-10 and DTIC-SLNs-11 corona and outer surface turns out to be fragile and leaky as compared to the DTIC-SLNs-9; which could result in less entrapment efficacy. It can be assumed that the number of micelles which can be migrated in the water phase would be much higher when phosphatidylcholine and Kolliphor^® were absent in formulation, which ultimately results in low encapsulation efficacy (%); This phenomenon can easily explain by observing higher particle size (821 ± 6.1nm &715 ± 7.36nm) and higher PDI (0.51 ± 0.023 & 0.27 ± 0.001) for DTIC-SLNs-10 & DTIC-SLNs-11.

Gold nanoparticles were coated with DTIC to learn more about drug localization inside prepared SLNs. According to Chaitali D. Dekiwadia et al. (2012), peptide-mediated cell penetration was studied using 13 nm gold nanoparticles, which are efficiently and selectively delivered into lysosomes with minimal cytotoxicity (45).

DTIC loaded colloidal gold nanoparticles develop a process of selection zone and were clustered in the shell area, as seen in Figure 5 . The evidence supports the localization hypothesis, implying that perhaps the formulation’s entrapment efficacy (%) was dependent on the correct use of phosphatidylcholine and Kolliphor^® in solid lipid nanoparticles. The gold nanoparticles collated DTIC composition also indicates that the nanoparticles’ laminated surface region facilitates steric hindrance.

Table 3 is indicating the permeation parameters for all 11 formulations and the free drug. The presence of phosphatidylcholine enhances the lipophilicity of the formulations; therefore, prepared SLNs can able to penetrate more abruptly though the hydrophobic-lipophilic membrane as compared to free drug (DTIC). The relationship of cumulative amounts of permeated DTIC for 24 h Q24(µg/cm³) and Lag time(min) in almost all the formulation shows a significant relationship. The Q24, which is known as the number of micrograms passing though 0.1 mm of the membrane in cumulative time, increased significantly. The formulae, which are comprising of enhanced phosphatidylcholine and Kolliphor^® concertation, are showing higher Q24 value. The DTIC-SLNs-8 shows the highest membrane permeability (275 ± 5.67µg/cm³).

The DTIC-SLNs-8 shows the highest permeability coefficient, known as the slope of the straight-line portion of the curve separated by the drug concentration formerly applied (0.5246 ± 0.039 cm/h) with the shortest lag time 40 ± 4.14min. Lower particle size, lower PDI, higher surface area and lower aggrupation’s are some of the attributions for enhance permeation and shortest lag time of DTIC-SLNs-8. It is very indispensable to have shortage lag time for any topical formulation as topical formulations are meant to be removed form skin mechanically by patients. Therefore, it is a prerequisite to deliver payload on the target site within a limited time. In order to conduct in vivo studies, formulations with higher drug entrapment near the core shell area and a faster drug distribution profile should be chosen. As a consequence, smaller particle size and PDI formulation known as DTIC-SLNs-8 was proposed for integration into several gel metrics. DTIC-SLNs-9 also had a good drug encapsulation efficacy (%), but the formulation PDI was higher than DTIC-SLNs-8. Therefore, DTIC-SLNs-8 was considered as an optimised batch.

According to RP. Thatipamula et al. (2011), the ultrasonication technique was used to prepare domperidone-loaded solid lipid nanoparticles and nanostructured lipid carriers. Stability tests were performed on these formulations for 40 days. The developed nanoparticles were fairly stable, with no significant (P<0.05) changes in particle size, zeta potential, PDI, or entrapment efficiency. Within nanoparticles, perfect ratios of trimyristin as a solid lipid, cetyl recinoleate as a liquid lipid, and a mixture of soy phosphatidylcholine (99%) and tween 80 as a surfactant were observed, resulting in an improved stability profile (46).

In the current research, the stability studies for DTIC-SLNs-8 were performed for 9 months. It was observed that zeta potential and polydispersity index not shown any big difference after 9-month stability studies, which indicates a good stability profile of the formulation. However, insignificant changes(p>0.05) of particle size increased from 146 ± 4.71nm to 154 ± 10.67nm, and a nearly 7.07% decrease of entrapment efficacy (%) was reported within nine months of stability studies ( Figure 6 ). The higher particle size was reported due to the evolution of Ostwald ripening effects between nanoparticles within nine months. Since the PDI of the DTIC-SLNs-8 did not alter dramatically, variations in electrostatic charges within the nanoparticles’ outer surface may be another explanation for the larger particle size. Furthermore, during Ostwald ripening, smaller particles diffuse across the outer surface of nanoparticles, creating larger particles. These observations are in accordance with those of other research published in the literature.

As per Usama A Fahmy et al. (2020) Flibanserin lipid carriers incorporated into 0.6% w/v gellan gum comprising nasal in-situ gel improve the drug bioavailability and drug delivery profile in brain (47).As a result, it’s reasonable to believe that the required concentration of gellan gum will result in good mucoadhesive properties. In the current experiment design gellan gum (0.01% w/v), a linear anionic polysaccharide, chitosan (0.5% w/v), a polycationic linear polysaccharide, polysarcosine (2.5%w/v), a cationic polypeptide was all incorporated to prepared multiple DTIC-SLNs-8 gel formulations. According to the diffusion studies, DTIC-SLNs-8 formulations has the best Q24, the shortest lag period, and the highest Kp value in Gellan gum (0.01% w/v) ( Tables 4 , 5 ).

The tumor weight ( Figure 7A ), tumor incidences were significantly reduced in DMBA group treated with DTIC-SLNs-8 with Gellan gum (0.01%w/v) ( Figure 7B ). In addition, DTIC-SLNs-8 treatment showed significant inhibition of tumor volume ( Figure 7C ) as well as inhibition of tumor multiplicity (number and size of tumors/rat) was markedly increased by DTIC-SLNs-8 treatment as compared to DMBA treated group ( Figure 7D ). Represent the invasion and metastatic condition of tumours in skin ( Figure 7E ). Showing that how DMBA +DTIC SLNs-8 inhibiting the invasion and metastatic condition of tumours in skin ( Figure 7F ). As we expected, and comparing to DMBA treated group, suggesting that DTIC-SLNs-8 inhibits DMBA-induced tumor growth.

Nuclear protein Ki67 has a close relationship with the cell cycle. It has been used to classify invasive skin cancer patients into prognostic groups with favourable and worse outcomes since it is a measure of cell proliferation. Its relationship to variations in gene expression has not yet been completely understood. In this work, slices taken from 21 invasive skin tumours that had been formalin-fixed and paraffin-embedded underwent Ki67 immunohistochemistry using the MIB-1 antibody. No immunostaining resulted in a score of zero, low immunopositivity (less than 10%), or strong immunoreactivity (more than 10%), respectively. The ki67 expression shown very higher( Figure 9A ), tumor ki-67 expression were significantly reduced in DMBA group treated with DTIC-SLNs-8 with Gellan gum (0.01%w/v) ( Figure 9B ). In addition, DTIC-SLNs-8 treatment showed significant downregulation of Ki-67 expression ( Figure 9C ) these findings suggesting that DTIC-SLNs-8 can inhibits DMBA-induced skin tumor growth in wistar rats.