Drug Integrating Amphiphilic Nano-Assemblies: 2. Spatiotemporal Distribution within Inflammation Sites

Abstract

The need for chronic systemic immunosuppression, which is associated with unavoidable side-effects, greatly limits the applicability of allogeneic cell transplantation for regenerative medicine applications including pancreatic islet cell transplantation to restore insulin production in type 1 diabetes (T1D). Cell transplantation in confined sites enables the localized delivery of anti-inflammatory and immunomodulatory drugs to prevent graft loss by innate and adaptive immunity, providing an opportunity to achieve local effects while minimizing unwanted systemic side effects. Nanoparticles can provide the means to achieve the needed localized and sustained drug delivery either by graft targeting or co-implantation. Here, we evaluated the potential of our versatile platform of drug-integrating amphiphilic nanomaterial assemblies (DIANAs) for targeted drug delivery to an inflamed site model relevant for islet transplantation. We tested either passive targeting of intravenous administered spherical nanomicelles (nMIC; 20-25 nm diameter) or co-implantation of elongated nanofibrils (nFIB; 5 nm diameter and >1 μm length). To assess the ability of nMIC and nFIB to target an inflamed graft site, we used a lipophilic fluorescent cargo (DiD and DiR) and evaluated the in vivo biodistribution and cellular uptake in the graft site and other organs, including draining and non-draining lymph nodes, after systemic administration (nMIC) and/or graft co-transplantation (nFIB) in mice. Localized inflammation was generated either by using an LPS injection or by using biomaterial-coated islet-like bead implantation in the subcutaneous site. A cell transplant inflammation model was used as well to test nMIC- and nFIB-targeted biodistribution. We found that nMIC can reach the inflamed site after systemic administration, while nFIB remains localized for several days after co-implantation. We confirmed that DIANAs are taken up by different immune cell populations responsible for graft inflammation. Therefore, DIANA is a useful approach for targeted and/or localized delivery of immunomodulatory drugs to decrease innate and adaptive immune responses that cause graft loss after transplantation of therapeutic cells.

Keywords: block-copolymers; cell transplantation; drug delivery; local immunomodulation; nanoparticles; self-assembling.

Scheme 1

Chemical composition of PEG₄₄-PPS₂₀ (A) and PEG₄₄-OES₅ (B) diblock-copolymers, and self-assembling into nanomicelles (nMIC) and nanofibrils (nFIB) with a cargo dye encapsulation.

Figure 1

In vitro uptake of nMIC and nFIB DIANAs into human islet cells. Optical fluorescent microscope images of human islets treated with nMIC-DiD or nFIB-DiD. Aliquots of 150 islet equivalents (IEQ) were treated with either (A) nMIC-DiD or (B) nFIB-DiD 100-folds diluted from the stocks to a concentration of 6.25 μg/mL per aliquot and incubated for 24 h. (C) Untreated islets were used as control. Images are shown at 10× magnification with DiD in magenta, and Hoechst stained cells are shown in blue. Scale bars = 200 μm.

Figure 2

Biodistribution of nMIC and nFIB DIANAs in model 1 of acute inflammation as revealed by whole-body imaging. Localized acute inflammation was obtained using subcutaneous injection of 25 μL of LPS from 1 mg/mL solution in saline in the right foot paw (RFP) of BALB/c mice (yellow arrows). Inflamed mice (two per condition) were treated with nMIC-DiD or nFIB-DiD via IV infusion and analyzed using whole-body imaging with IVIS spectrum at day 1 (A), 4 (B), and 6 (C). Radiant efficiency color scales: min = 6.0 × 10⁹, max = 2.0 × 10¹⁰. (D) In vivo time-dependent DiD fluorescence intensity for the right- (ipsilateral) and left foot paws (LFP, contralateral) quantified using a Living Image Software with the region of interest (ROI) tool (baseline-normalized compared to the untreated mouse). Asterisks indicate statistically significant differences between the RFP and LFP (n = 2 independent animals): * p < 0.05, ** p < 0.01. (E) Ex vivo IVIS spectrum images of major organs (lungs, spleen, pancreas, kidneys, and liver) extracted on day 7 after infusion. Radiant efficiency color scales: min = 2.5 × 10⁹, max = 5.0 × 10⁹.

Figure 3

Biodistribution of nMIC-DiD in model 2 of acute inflammation. Localized acute inflammation was obtained using subcutaneous implantation (SC) of either PEG coated beads (CB, blue) or of 25 μL of LPS (red) into a biological scaffold (BSc). (A) Sample image of the polystyrene beads coated with crosslinked PEG-maleimide used. Magnification = 10×. (B) In vivo biodistribution observed via live optical whole-body imaging of C57BL/6 mice implanted with CB (blue circles), LPS (red circles), or not-implanted (Not impl.; black circles) and treated with IV infusion of 50 μL of nMIC-DiD. Selected time points are shown (1, 4, 7, and 22 days). Radiant efficiency color scales: min = 1.0 × 10⁹, max = 6.0 × 10⁹. (C) Time-profile of the nMIC-DiD fluorescence intensity quantified using the left inguinal area as region of interest (ROI) in mice with the CB implant (blue circles), LPS implant (red square), and no implant (black triangle) from day 1 to 22. (D,E) Distribution of nMIC-DiD determined ex vivo at day 1 (D) and 22 (E) in the SC implant site (containing either CB or LPS within a BSc), left inguinal lymph node (ipsilateral LN), right brachial lymph node (contralateral LN), heart, lungs, liver, pancreas, spleen, and kidneys. Explanted organs were imaged using the IVIS spectrum system and their fluorescence intensity was quantified using the Living Imaging Software and the ROI method. Data are expressed as mean +/− SD. A ★ symbol indicates significance between CB + nMIC-DiD and nMIC-DiD p < 0.05; ♦ symbol indicates significance between LPS + nMIC-DiD and MIC-DiD p < 0.05; * indicates significance between SC tissue compared to other tissues p < 0.05 (n = 3 independent animals).

Figure 4

In vivo cellular uptake of nMIC-DiD in model 2 of acute inflammation. At days 1 (A) and 22 (B) post-graft incorporation, T cells (CD3⁺), B cells (CD19⁺), M1 macrophages (F4/80⁺MHC-II⁺CD206⁻), M2 macrophages (F4/80⁺MHC-II⁺CD206⁺), dendritic cells (DCs; F4/80⁻CD11c⁺), and neutrophils (CD3⁻CD19⁻Ly6G⁺) were quantified using flow cytometry for DiD-nMIC uptake in the SC graft. Data shown as percentage of live, CD45, and DiD positive cells (mean ± SD for n = 3 independent animals with CB + nMIC-DiD in blue, LPS + nMIC-DiD in red, and nMIC-DiD control in black). Data are expressed as mean +/− SD. A blue asterisk (*) symbol indicates a significant difference between neutrophils compared to other cells in CB + nMIC-DiD condition; a red asterisk symbol indicates a significant difference between neutrophils compared to other cells in LPS + nMIC-DiD condition; a black asterisk symbol indicates a significant difference between neutrophils compared to other cells in nMIC-DiD condition; a blue circle (•) symbol indicates a significance between T cells compared to other cells in CB + nMIC-DiD condition; a red circle symbol indicates a significance between T cells compared to other cells in LPS+DiD-nMIC condition ** p < 0.01, *** p < 0.001, **** p < 0.0001, as well as for circle symbols.

Figure 5

Biodistribution of nFIB-DiD in model 2 of acute inflammation. Localized acute inflammation was obtained using SC implantation of either CB (blue) or 25 μL of LPS (red) into a BSc in hairless SKH-1 mice. Five μL of nFIB-DiD was mixed with CB or LPS and implanted together in the SC BSc. (A) Biodistribution of nFIB-DiD co-implanted either with CB or LPS at day 1 after implantation as observed using in vivo whole-body imaging with IVIS spectrum. Radiant efficiency color scale: min = 1.0 × 10¹⁰, max = 1.0 × 10¹¹. (B) Time-profile of nFIB-DiD fluorescence intensity quantified using the right inguinal area as ROI in mice with the either the CB (blue circle) or the LPS implant (red circle) from day 1 to 21. Stars indicate statistically significant differences versus time 0 in the CB and LPS implants (blue and red, respectively; two-way ANOVA with Tukey’s post hoc testing; ** p < 0.01, *** p < 0.001, **** p < 0.0001. (C) Distribution of nFIB-DiD determined ex vivo at day 21 in the SC implant site containing either CB (blue) or LPS (red) within a BSc. Explanted organs were imaged using the IVIS spectrum system and their fluorescence intensity was quantified using the Living Imaging Software and the ROI method. Total and average radiant efficiency corrected via subtraction of control background mouse (n = 3 independent animals; data shown as mean ± SD).

Figure 6

In vivo cellular uptake of nFIB-DiD in model 2 of acute inflammation. At days 1 (A) and 21 (B) post-graft incorporation, B cells (CD19⁺), T cells (CD3⁺), DCs (F4/80⁻CD11c⁺), neutrophils (CD3⁻CD19⁻Ly6G⁺), M1 macrophages (F4/80⁺MHC-II⁺CD206⁻), and M2 macrophages (F4/80⁺MHC-II⁺CD206⁺) were quantified using flow cytometry for nFIB-DiD uptake in the SC graft. In the graphs on the left (A,B), data are shown as a percentage of live, CD45⁺, and DiD positive cells (mean ± SD for n = 3 independent animals with CB + nFIB-DiD in blue, LPS + nFIB-DiD in red, and nFIB-DiD control in black). In the right plots (C,D), data shown are % DiD positive cells of live CD45⁺ cells (colored scale) and immune cell fraction (dot radius).

Figure 7

nFIB-DiR biodistribution in syngeneic pancreatic islet transplantation (model 3). (A–C) Illustration of the transplant procedure: the left gonadal fat pad was exteriorized and spread over a sterile field (A), islets and 15 μL of nFIB-DiR (blue) were immobilized on the EFP using a BSc (not visible) (B), and the EFP was folded over the islets/nFIB BSc to be placed back into the abdominal cavity (C). (D) Spatiotemporal localization of nFIB in the site of islet implantation monitored at post operatory days 3, 4, 5, 7, and 14 (panels 1 to 5) via whole-body imaging with IVIS spectrum using the near infrared DiR fluorescence emission. Mice implanted only with islets were used as negative controls. Radiant efficiency color scale: min = 0.8 × 10⁸, max = 5.0 × 10⁸. (E) ROI analysis of the implanted EFPs at the different time points normalized with the background (p value > 0.05, n = 2 independent animals; data shown as mean ± SD). (F) Functionality of the transplanted islets as monitored via blood glucose level shows no impairment in the presence of nFIB-DiR (blue and cyan vs. black lines) indicating lack of toxicity (n = 2 independent animals).

Figure 8

nMIC-DiD biodistribution in allogeneic pancreatic islet transplantations (model 3). C57BL/6 mice with highly compromised EFP islet transplants (nearing full rejection and no longer functional) were treated with 50 μL of nMIC-DiD via IV infusion. (A–E) Ex vivo IVIS imaging of explanted organs from control mice that were not transplanted and infused (A), mice bearing an islet transplant at day 1 (B) and 4 (C) after nMIC-DiD IV infusion, and mice that did not receive a transplant but received IV infusion of nMIC-DiD at day 1 (D) and 4 after infusion (E). Radiant efficiency color scale: min = 0.6 × 10¹⁰, max = 1.8 × 10¹⁰. (F) Quantification of DiD fluorescence of the explanted organs normalized by mass weight (mg) using ROI analysis with IVIS spectrum from transplanted and non-transplanted mice (dark and light blue bars, respectively). The asterisks in red indicate statistically significant difference between each organ of the transplanted mice and the corresponding organ of the non-transplanted mice. Dark-blue and sky-blue asterisks indicate that the presence of nMIC in the lungs is significantly higher than in all the other organs analyzed in transplanted and non-transplanted mice, respectively: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 (2 separate experiments with n = 3 animals per experiment).