(168t) Multifunctional Nanodelivery System for Prostate Cancer Treatment

Prostate cancer (PC) is the most lethal cancer type causing more than 33,000 deaths of American men annually.¹ The first line therapy based on androgen ablation has only been providing palliative benefit (5-year overall survival rate ~98%) rather than desired curative outcomes, eventually resulting in development of aggressive metastatic “castration resistant prostate cancer” (CRPC) in 20% of patients with median survival rate of 16 to 18 months.²

Enzalutamide (Enz), an orally administered androgen receptor (AR) inhibitor, is commonly used for treatment of metastatic CRPC targeting AR signaling axis, however, the limited bioavailability of Enz lead to high dose exposure resulting in systemic side effects. ^3,4 The recent application of gold nanoparticles for photothermal ablation of prostate tumors can be considered as a promising strategy with reduced side effects, however, this method alone does not eliminate PC recurrence.⁵ Therefore, there is a critical need for alternative combinatorial treatment strategies and therapeutic interventions for metastatic PC.

MicroRNAs (miRNAs), a class of small non-coding regulatory RNAs, can be used as an alternative therapeutic intervention targeting CRPCs. Particularly, miR-149 has been shown to be effective in decreasing PC tumor growth in mice models via downregulation of AR signaling axis in our previous work.⁶ However, application of miRNA therapy is limited by challenges related to targeted delivery, stability, transport, diffusion, cellular entry, endosomal escape, efficient release and therapeutic activity. Therefore, an ideal delivery system combining multiple functionalities holds potential to overcome current limitations for efficient miR-149 delivery and direct CRPC.

In this study, we developed gold nanoparticles and cell penetrating/fusogenic peptide-based multifunctional nanoscale delivery system (MNDS) that can provide (i) miR-149 and Enz co-loading (ii) targeted co-delivery, and (iii) localized photothermal therapy to effectively modulate tumor microenvironment, inhibit AR signaling axis, and promote Enz bioavailability and activity. For this purpose, MNDS were prepared via following steps described in our previous works.^7–9 Briefly, AuNPs constituting the core of the MNDS were prepared using traditional citrate reduction. Thiol modified miR-149 was attached to the surface of AuNPs via gold-sulfur interactions to obtain negatively charged AuNP/miR-149 particles. At the same time, carboxyl groups of TAT-HA2 peptide was crosslinked to amine groups of PSMA targeting GTI peptide using EDC-NHS chemistry to obtain TAT-HA2-GTI conjugate. TAT protein transduction domain (PTD) binds to the cell surface and penetrates the membrane via lipid raft-dependent macropinocytosis. Endosomal escape and transduction of fusion peptide are enhanced by the HA2 domain, which is a pH-sensitive lipid membrane destabilizing sequence. GTI domain provides PSMA specific cell targeting. Enz was encapsulated into TAT-HA2-GTI peptide via hydrophobic interactions and self-assembly. Finally, negatively charged AuNP/miR-149 was covered by positively charged Enz/TAT-HA2-GTI layers at certain ratios via electrostatic interactions.

The change in size and zeta potential of AuNPs were determined by Dynamic Light Scattering (DLS) while the red shift in specific wavelength of bare AuNPs was used to verify the surface modification of AuNPs via UV-vis spectrophotometer after each step. The miR-149 condensation and MNDS formation long with miR-149 protection was visualized using gel electrophoresis.^7–9 The MNDS stability in terms of size and zeta potential in serum-containing cell culture media was observed by DLS and UV-vis.^7–9 To test the in vitro therapeutic efficaicy of the MNDS we used human PC cell lines (LNCaP, PC3, DU145, 22Rv1) and the normal epithelial prostate cells (PrEc) as described in our previous work.¹⁰ The targeting and cellular uptake of MNDS was evaluated through the Inductively Coupled Plasma Mass Spectroscopy (ICP-MS) analysis.^7,8 The intracellular distribution and endosomal escape capabilities of the MNDS was evaluated by confocal microscopy as described previously.^7,8 To evaluate the in vitro efficiency of the MNDS to kill PC cells, cell viability was assessed using different MNDS doses coupled with near infrared (NIR) laser stimulation at 808 nm (1-4 W with 4 mm spot size) at different time periods. The cells transfected by MNDS in presence and absence of NIR laser exposure were assessed in terms of downregulation of AR signaling via determination of endogenous prostate specific antigen (PSA) expression at mRNA level by qRT-PCR and at protein level by PSA ELISA.¹¹ The apoptotic cells were also detected by using the cell death detection ELISA kit and Annexin V-FITC staining.¹¹

Our results showed that miR-149 expression is significantly decreased in PC tissues and cell lines^6,12. We demonstrated that miR-149 directly targets AR and SREBP1 consequently causing significant downregulation of HMGCR, HMGCs and SCARB1 expression, affecting intracellular cholesterol levels and precursor of DHT synthesis. We successfully co-loaded miR-149 to the MNDS along with Enz. The nanoscale systems demonstrated stability against serum proteins and RNaze enzymes. The restoration of miR-149, in combination with Enz and photothermal therapy via MNDS, enhanced downregulation of AR signaling and enhanced Enz sensitivity resulting improving therapeutic outcome in in vitro PC cells. In conclusion, the nanodelivery combination therapies for miR-149/Enz and photothermal stimulation demonstrated potential for future in vivo studies and PC treatment.

References:

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