Microparticle Depots for Controlled and Sustained Release of Endosomolytic Nanoparticles

Abstract

Introduction: Nucleic acids have gained recognition as promising immunomodulatory therapeutics. However, their potential is limited by several drug delivery barriers, and there is a need for technologies that enhance intracellular delivery of nucleic acid drugs. Furthermore, controlled and sustained release is a significant concern, as the kinetics and localization of immunomodulators can influence resultant immune responses. Here, we describe the design and initial evaluation of poly(lactic-co-glycolic) acid (PLGA) microparticle (MP) depots for enhanced retention and sustained release of endosomolytic nanoparticles that enable the cytosolic delivery of nucleic acids.

Methods: Endosomolytic p[DMAEMA]_10kD-bl-[PAA_0.3-co-DMAEMA_0.3-co-BMA_0.4]_25kD diblock copolymers were synthesized by reversible addition-fragmentation chain transfer polymerization. Polymers were electrostatically complexed with nucleic acids and resultant nanoparticles (NPs) were encapsulated in PLGA MPs. To modulate release kinetics, ammonium bicarbonate was added as a porogen. Release profiles were quantified in vitro and in vivo via quantification of fluorescently-labeled nucleic acid. Bioactivity of released NPs was assessed using small interfering RNA (siRNA) targeting luciferase as a representative nucleic acid cargo. MPs were incubated with luciferase-expressing 4T1 (4T1-LUC) breast cancer cells in vitro or administered intratumorally to 4T1-LUC breast tumors, and silencing via RNA interference was quantified via longitudinal luminescence imaging.

Results: Endosomolytic NPs complexed to siRNA were effectively loaded into PLGA MPs and release kinetics could be modulated in vitro and in vivo via control of MP porosity, with porous MPs exhibiting faster cargo release. In vitro, release of NPs from porous MP depots enabled sustained luciferase knockdown in 4T1 breast cancer cells over a five-day treatment period. Administered intratumorally, MPs prolonged the retention of nucleic acid within the injected tumor, resulting in enhanced and sustained silencing of luciferase relative to a single bolus administration of NPs at an equivalent dose.

Conclusion: This work highlights the potential of PLGA MP depots as a platform for local release of endosomolytic polymer NPs that enhance the cytosolic delivery of nucleic acid therapeutics.

Keywords: Biomaterial; Drug delivery depot; Endosomal escape; Immunotherapy; Intratumoral; Local delivery; Nucleic acid therapeutics; PLGA; RNA interference.

Figure 1

PLGA microparticle depots for controlled release of endosomolytic nanoparticles. (a) PLGA MP depots mediate local nanoparticle release and subsequent intracellular delivery of nucleic acid to local cell populations. (b) Structure and composition of the endosomolytic diblock copolymers used for cytosolic nucleic acid delivery. (c) Representative scanning electron microscopy (SEM) images of nonporous microparticles (left) and porous microparticles (right). Scale: 3 µm.

Figure 2

In vitro characterization of PLGA microparticle depots. (a) Particle size distribution of nonporous and porous MPs determined by laser diffraction particle sizing. (b) In vitro release profiles of NPs from porous and nonporous MP depots over a 15 day period. (c) Longitudinal analysis of luciferase silencing in 4T1-LUC breast cancer cells treated with a single administration of either free NPs or porous MPs. The NP treatments were removed after 24 h, while MPs were left in coculture with the cells throughout the experiment to mimic biological residence. Luminescent signal for each treatment group was normalized to that of an analogous treatment containing scrambled negative control RNA substituted for luciferase siRNA.

Figure 3

In vivo retention and release of nanoparticles from PLGA microparticles. In vivo analysis of injection site localization of free NPs, nonporous MP depots, and porous MP depots in BALB/c mice. (a) Relative fluorescence of Alexa Fluor® 647(A647)-labelled dsDNA cargo injected subcutaneously and monitored over 56 days. (b) Relative fluorescence of A647-labelled dsDNA cargo, releasing from an intratumoral injection site over 14 days. The fluorescent efficiency of each mouse was captured by IVIS imaging and was normalized to the respective initial (day 0) fluorescence. (c) Representative IVIS images of mice bearing subcutaneously administered particles containing fluorescent dsDNA (Red). (d) Representative IVIS images of the mice treated intratumorally with particles containing fluorescent dsDNA (Red).

Figure 4

In vivo activity of PLGA microparticle depots for siRNA delivery. In vivo activity of free NP and porous MPs delivering Alexa Fluor® 647 siRNA cargo was investigated in an orthotopic 4T1-LUC breast cancer model. (a) Fluorescent (top) and overlaid fluorescent and bright field (bottom) images of cyrosections of tumor tissue following intratumoral injection of porous MPs. Scale: 75 µm. (b) Representative IVIS images of mice bearing luciferase-expressing 4T1-LUC cells (Blue), treated intratumorally with fluorescent RNA (Red). (c) Longitudinal analysis of luciferase silencing in a 4T1-LUC breast cancer tumor model treated with a single intratumoral injection of either free NPs or porous MPs. Luminescent signal for each treatment group was normalized to that of an analogous treatment containing scrambled negative control RNA substituted for luciferase siRNA.