Laser ablation for pharmaceutical nanoformulations: Multi-drug nanoencapsulation and theranostics for HIV

Abstract

We introduce the use of laser ablation to develop a multi-drug encapsulating theranostic nanoformulation for HIV-1 antiretroviral therapy. Laser ablated nanoformulations of ritonavir, atazanavir, and curcumin, a natural product that has both optical imaging and pharmacologic properties, were produced in an aqueous media containing Pluronic® F127. Cellular uptake was confirmed with the curcumin fluorescence signal localized in the cytoplasm. Formulations produced with F127 had improved water dispersibility, are ultrasmall in size (20-25 nm), exhibit enhanced cellular uptake in microglia, improve blood-brain barrier (BBB) crossing in an in vitro BBB model, and reduce viral p24 by 36 fold compared to formulations made without F127. This work demonstrates that these ultrasmall femtosecond laser-ablated nanoparticles are effective in delivering drugs across the BBB for brain therapy and show promise as an effective method to formulate nanoparticles for brain theranostics, reducing the need for organic solvents during preparation.

Keywords: Antiretroviral therapy; Blood–brain barrier crossing; Laser ablation; Multi-drug ablation; Multi-drug encapsulation.

Figure 1.

Preparation of aqueous laser ablated nanoparticles. (a) experimental setup; (b) schematic of NP obtained after aqueous laser ablation

Figure 2.

Particle size distribution of laser ablated ART NPs determined by DLS.

Figure 3.

Representative TEM images of laser ablated NPs: Produced in water; (a) ATV; (b) RTV; (c) Cur, (d) ARC. Produced in water containing F127; (e) FATV; (f) FRTV; (g) FCur; (h) FARC.

Figure 4.

Absorption (a) and emission (b) spectra of laser ablated ANT, ARA, FANT and FARA NPs in water. Absorption (c) and emission (d) spectra of laser ablated FCur and FARC NPs in water.

Figure 5.

Cellular uptake of laser ablated ART NPs in CHME-5/HIV cells. (a) FCur; (b) FARC; (c) Cur; and (d) ARC. The cells were incubated for 2 h at 37°C. Left to right: bright field; blue is DAPI staining; green is curcumin channel; image overlay. Scale bar represents 100μm.

Figure 6.

Cell viability of laser ablated ART NPs in (a) CHME cells and (b) CHME-5/HIV cells.

Figure 7.

(a) Schematic of in vitro BBB model (b) Ability of laser ablated ART NPs to cross the BBB.

Figure 8.

(a) Effect of NPs on LTR gene expression. Cells infected with HIV-1 were treated with the NPs. RNA was extracted and quantified by RT-qPCR using primers specific for the LTR region of the HIV-1 genome. (b) Effect of laser ablated ART NPs on HIV-1 replication in THP-1/HIV cells. FARC NPs suppress HIV-1 replication.