Ionic Driven Embedment of Hyaluronic Acid Coated Liposomes in Polyelectrolyte Multilayer Films for Local Therapeutic Delivery

Abstract

The ability to control the spatial distribution and temporal release of a therapeutic remains a central challenge for biomedical research. Here, we report the development and optimization of a novel substrate mediated therapeutic delivery system comprising of hyaluronic acid covalently functionalized liposomes (HALNPs) embedded into polyelectrolyte multilayer (PEM) platform via ionic stabilization. The PEM platform was constructed from sequential deposition of Poly-L-Lysine (PLL) and Poly(Sodium styrene sulfonate) (SPS) "(PLL/SPS)4.5" followed by adsorption of anionic HALNPs. An adsorption affinity assay and saturation curve illustrated the preferential HALNP deposition density for precise therapeutic loading. (PLL/SPS)2.5 capping layer on top of the deposited HALNP monolayer further facilitated complete nanoparticle immobilization, cell adhesion, and provided nanoparticle confinement for controlled linear release profiles of the nanocarrier and encapsulated cargo. To our knowledge, this is the first study to demonstrate the successful embedment of a translatable lipid based nanocarrier into a substrate that allows for temporal and spatial release of both hydrophobic and hydrophilic drugs. Specifically, we have utilized our platform to deliver chemotherapeutic drug Doxorubicin from PEM confined HALNPs. Overall, we believe the development of our HALNP embedded PEM system is significant and will catalyze the usage of substrate mediated delivery platforms in biomedical applications.

Figure 1. Fabrication overview, deposition validation, and characterization of the Polyelectrolyte Multilayer (PEM) base structure (PLL/SPS)_4.5.

(a) Fabrication Schematic for the PEM base structure utilizing layer-by-layer adsorption of PLL and SPS. Chem draw and powerpoint was used to create the drawings. (b) UV Spectrum Analysis of Bilayer Deposition. (c) UV Absorbance Readings (220 nm, characteristic for SPS) as a function of bilayer addition to validate successful base construction. (d) AFM of the (PLL/SPS)_4.5 base platform via height (i) and phase contrast (ii) analysis to visually demonstrate a uniform surface ideal for nanoparticle deposition. Scale bar for AFM is 1 μm. Figure drawn by Stephen L Hayward.

Figure 2. Ionic driven embedment of Hyaluronic Acid Coated Lipid Nanoparticles (HALNPs) in the PEM Platform (HALNP-PEM).

(a) Nanoparticle embedded PEM platform overview and proposed hypothesis of action with the (PLL/SPS)_2.5 capping layer. Chem draw and powerpoint was used to create the drawings. Figure drawn by Stephen L Hayward. (b) Preferential deposition of HALNPs on PLL over SPS topped surfaces. Capillary Force Lithography (CFL) was used to create PLL patterns on (PLL/SPS)₄ and to demonstrate the level of spatial control for nanoparticle adsorption. Fluorescent Recovery after Photo bleaching (FRAP) Analysis pre and post the addition of the capping layer for 10 minutes (c), and with capping layer utilizing CFL for 30 minutes (d). The yellow arrows are pointing to particles that have moved during the experimental time lapse.

Figure 3. Quantitative analysis of HALNP Loading in the PEM system and subsequent release profile.

(a) Affinity assay for HALNP absorption on (PLL/SPS)_4.5 and (PLL/SPS)₄ substrates to determine the optimum conditions for nanoparticle deposition. (b) Saturation Curve analysis of Total Lipid Loaded on (PLL/SPS)_4.5 per square centimeter as a function of total lipid added during the three hour incubation time. (c) HALNP nanocarrier release profile from the non-capped [(PLL/SPS)_4.5(HALNP)] and capped [(PLL/SPS)_4.5(HALNP)(PLL/SPS)_2.5] HALNP-PEM platforms.

Figure 4. 21MT-1 metastatic breast cancer cell uptake of fluorescently conjugated HALNPs from the embedded PEM system with capping layer as a function of time [(PLL/SPS)_4.5(HALNP)(PLL/SPS)_2.5].

Flow Cytometry Analysis of (a) percent cell population FITC positive and (b) per cell fluorescence directly comparing nanoparticle uptake between the HALNP-PEM and HALNP bolus systems (*P < 0.05, **P < 0.005; n = 3; ^#denotes significance between a specific sample type and the preceding time point following the same significance scale as the stars). (c) Phase Contrast and Fluorescent Microscopy visual investigation of nanoparticle intracellular delivery on the HALNP-PEM platform.

Figure 5. Doxorubicin potency assay comparison between Free Dox, DOX encapsulated in HALNPs (HALNPDOX) in the bolus form, and HALNPDOX nanoparticles embedded into the PEM platform (HALNPDOX-PEM).

Standard MTT protocol was used to determine the % viable cells at 24 hours.

Figure 6. DOX release profile from the non-capped [(PLL/SPS)_4.5(HALNP)] and capped [(PLL/SPS)_4.5(HALNP)(PLL/SPS)_2.5] HALNP-PEM platforms as a function of time utilizing the chemotherapeutics natural fluorescence for quantification.

Figure 7. Local delivery of patterned HALNPs via the HALNP-PEM platform.

Phase contrast and fluorescent microscopy images of 21MT-1 metastatic breast cancer cells adhered to the HALNP-PEM system to visually probe both the temporal and spatial release of the HALNP nanocarrier. Capillary force lithography (CFL) was used to pattern PLL and create long range order (a) fluorescently tagged HALNP and (b) HALNPDOX patterns via preferential nanoparticle adsorption.