Enhanced drug delivery and wound healing potential of berberine-loaded chitosan-alginate nanocomposite gel: characterization and in vivo assessment

Abstract

Berberine-encapsulated polyelectrolyte nanocomposite (BR-PolyET-NC) gel was developed as a long-acting improved wound healing therapy. BR-PolyET-NC was developed using an ionic gelation/complexation method and thereafter loaded into Carbopol gel. Formulation was optimized using Design-Expert® software implementing a three-level, three-factor Box Behnken design (BBD). The concentrations of polymers, namely, chitosan and alginate, and calcium chloride were investigated based on particle size and %EE. Moreover, formulation characterized in vitro for biopharmaceutical performances and their wound healing potency was evaluated in vivo in adult BALB/c mice. The particle distribution analysis showed a nanocomposite size of 71 ± 3.5 nm, polydispersity index (PDI) of 0.45, ζ-potential of +22 mV, BR entrapment of 91 ± 1.6%, and loading efficiency of 12.5 ± 0.91%. Percentage drug release was recorded as 89.50 ± 6.9% with pH 6.8, thereby simulating the wound microenvironment. The in vitro investigation of the nanocomposite gel revealed uniform consistency, well spreadability, and extrudability, which are ideal for topical wound use. The analytical estimation executed using FT-IR, DSC, and X-ray diffraction (XRD) indicated successful formulation with no drug excipients and without the amorphous state. The colony count of microbes was greatly reduced in the BR-PolyET-NC treated group on the 15th day from up to 6 CFU compared to 20 CFU observed in the BR gel treated group. The numbers of monocytes and lymphocytes counts were significantly reduced following healing progression, which reached to a peak level and vanished on the 15th day. The observed experimental characterization and in vivo study indicated the effectiveness of the developed BR-PolyET-NC gel toward wound closure and healing process, and it was found that >99% of the wound closed by 15th day, stimulated via various anti-inflammatory and angiogenic factors.

Keywords: alginate; berberine; chitosan; nanocomposite; nanomedicine; nanotechnology; polymer; wound healing.

Figure 1

Response surface curve (A–E) exploring the impact of independent variables on dependent variables in the preparation of NPs. Two dimensional contour curve (F–J) exhibiting the influence independent variables have on dependent variables of NPs.

Figure 2

Histogram showing particle size distribution curve (A); TEM image of BR–PolyET–NC (B); SEM image of BR–PolyET–NC gel (C).

Figure 3

Percentage of BR release from BR–PolyET–NC and BR-dispersion at pH 6.8.

Figure 4

(A) XRD, and (B) FTIR of berberine nanocomposite.

Figure 5

Viscosity (Pa.s) versus shear rate (1/s) profile of BR-PolyET-NC gel.

Figure 6

(A) Time dependent (0, 5, 10, and 15 days) healing of the wound in Balb C mice following treatment with BR–PolyET–NC, BR gel, blank gel, and positive control (untreated). (B) Wound closure measured on days 5, 10, and 15 after incision made on skin following treatment with BR–NP gel, BR gel, and blank and positive control. (C) Microbial colony count at the wound site. Data were expressed as mean ± SD (mean ± SD = 3). Observations among the group were statistically compared using a one-way ANOVA and Tukey’s multiple comparison test. The level of significance; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 when compared to different groups.

Figure 7

Histopathological examination of various treatment groups showing the changes from the 5th day to the 15th day of therapy in BALB/c mice: BR–PolyET–NC gel treated group; BR gel treated; blank gel treatment group; and untreated or positive control and negative or normal at the end of the 15th day. The BR–PolyET–NC gel treated group stated the great influence in wound healing capability as demarcated by keratinization, formation of hair follicles, or skin appendages. The blank gel and BR gel groups indicated acanthosis and follicular hyperkeratosis and inflammatory infiltrates in dermal tissues. The connective fibrous tissue marks the existence of inflammatory infiltrates. Scale bar: 400 μm.