Dacarbazine-encapsulated solid lipid nanoparticles for skin cancer: physical characterization, stability, in-vivo activity, histopathology, and immunohistochemistry

Abstract

Background: This study examined the use of solid lipid nanoparticles (SLNs) to administer Dacarbazine (DTIC) to skin melanoma cells with minimal adverse effects. Melanoma is a tricky skin cancer to cure, and standard chemotherapy has many negative effects. Encapsulating DTIC in SLNs may allow the drug to target melanoma cells without harming healthy cells. The study developed and tested DTIC-loaded SLNs for skin melanoma treatment.

Methods: This study encapsulated Dacarbazine (DTIC) in solid lipid nanoparticles (SLNs). SLNs with reversed micelles were produced utilizing specified ratios of the surfactant Kolliphor^® P188 and phosphatidylcholine. To track SLN drug localisation, gold nanoparticles were conjugated to the DTIC. Nanoparticle size and form were examined using DLS and TEM. These approaches ensured SLNs had the correct size and shape for drug delivery.

Significant findings: In the study, various parameters of the developed solid lipid nanoparticles (SLNs) were evaluated, including particle size, zeta potential, polydispersity index (PDI), entrapment efficacy, and cumulative drug permeation. The values for these parameters varied across the different formulations, with particle size ranging from 146 ± 4.71 nm to 715 ± 7.36 nm, zeta potential from -12.45 ± 2.78 mV to -30.78 ± 2.83 mV, PDI from 0.17 ± 0.013 to 0.51 ± 0.023, entrapment efficacy from 37.78 ± 2.78% to 87.45 ± 4.78%, and cumulative drug permeation from 117 ± 4.77 μg/cm² to 275 ± 5.67 μg/cm². To determine the optimal anti-cancer formulation, the DTIC-SLNs-8 nanoparticles were mixed with an optimized concentration of Gellan gum (0.01% w/v) and applied to DMBA-induced skin tumors in rats for six weeks, twice daily. Histopathology demonstrated that DTIC-SLNs-8-treated rats had less keratosis, inflammatory responses, and angiogenesis than free DTIC-treated rats. The development of SLNs may be a promising approach for melanoma treatment due to their improved drug retention over the skin. The optimised anti-cancer formulation DTIC-SLNs-8 showed improved efficacy with minimal side effects as compared to free DTIC.

Keywords: cancer; ehrlich ascetic carcinoma; melanoma; solid lipid nanoparticles; wistar rats.

Figure 1

Phosphatidylcholine (mg) and Kolliphor^® P188 (w/v%) impact on encapsulation efficacy percentage were correlated.

Figure 2

Transmission electron microscopy (TEM) images of the prepared DTIC-SLNs-9 at 100 nm scale (A) and 200 nm scale (B). The histogram of DTIC-SLNs diameters indicating 188.70nm as average diameter with 34.66nm standard deviation (C). The mean surface area of DTIC-SLNs was found to be 8693.63 nm³ with a standard deviation of 1600.66 nm³ (D).

Figure 3

Mechanism and Formation of Micelles while formulating DTIC-SLNs.

Figure 4

TEM image of DTIC-SLNs-10; without Kolliphor^® (A) with 3D surface morphology (B). TEM image of DTIC-SLNs-11; without phosphatidylcholine (C) with 3D surface morphology (D).

Figure 5

Gold nanoparticles coating with DTIC to check drug localization within SLNs.

Figure 6

Nine month Stability studies results compilation for DTIC-SLNs-8; (A) Entrapment efficacy (%) and particle size (nm) after nine months (B) Polydispersity index (PI) after nine months (C) Zeta potential (mV) after nine months.

Figure 7

DTIC-SLNs-8 inhibits tDMBA-induced skin tumor occurrence, multiplicity, volume and weight. Representative bar graph are shown the effect of DTIC-SLNs-8 on DMBA-induced skin tumorigenesis; (A) the change of tumor weight of rats in every two weeks until the end of experiment period i.e. 18 weeks, (B) percentage of tumor incidence of rats thoughout the duration of experiment, (C) percent inhibition of tumor multiplicity with respect to DMBA alone treated group, (D) average tumor volume of all experimental groups, (E) Vehicle with DMBA tumor image (F) DMBA +DTIC-SLNs-8 tumor image (G) DMBA + DTIC tumor image. Control, acetone (vehicle) treated group; DMBA, DMBA/TPA treated group; DMBA+DTIC-SLNs-8, DMBA/TPA-treated rats with DTIC-SLNs-8 with Gellan gum (0.01%w/v); DMBA+DTIC, DMBA/TPA-treated rats with DTIC with Gellan gum (0.01%w/v). Each data point is represented as mean ± SD of 10 rats in each group. Statistical analyses were performed with Student’s t-test. *p < 0.01 compared with DMBA alone treated group. **P ≤ 0.01. represent significant different than disease control group.

Figure 8

Histology (hematoxylin-eosin (H&E) staining) of the skin of hairless rats after treatment with DMBA+ DTIC-SLNs-8 and DMBA+ DTIC (0.01%w/v). (A) (0.01%w/v) Group; (B) DMBA+ DTIC-SLNs-8 Group; (C) DMBA+ DTIC Group. Each specimen was subjected to H&E staining and photographed at a magnification of 400x. Scale bar = 100 µm. (A) Arrow represents Squamous cell carcinomas with large nuclei and high mitotic figure & inflammation: H&E x400. (B) Squamous cell carcinomas with very minure nuclei and minure mitotic figure. (C) Squamous cell carcinomas with moderate nuclei and moderate mitotic figure H&E x400.

Figure 9

(A) represents overexpression of Ki-67 in DMBA treated group. (B) indicating DMBA+DTIC-SLNs-8 decreases proliferative cells in the tumor section in DMBA-induced skin tumors. (C) representing the moderate downregulation of Ki-67 expression. Representative images micrographs of Ki-67 staining (nuclei stained with brown color) in the tumor section of experimental groups are shown. Bar graph is shown the percentage of proliferative cells by counting Ki-67 positive cells in 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. vehicle Control treated group; DMBA treated group; DMBA+ DTIC-SLNs-8 with Gellan gum (0.01%w/v); DMBA+DTIC with Gellan gum (0.01%w/v) Statistical analyses were performed with Student’s t-test *p < 0.01 compared with DMBA alone treated group.