Enhancing Vascularization through the Controlled Release of Platelet

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Enhancing Vascularization through the Controlled Release of Platelet-Derived Growth Factor-BB Silvia Minardi, Laura Pandolfi, Francesca Taraballi, Xin Wang, Enrica De Rosa, Zachary D Mills, Xuewu Liu, Mauro Ferrari, and Ennio Tasciotti ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b13760 • Publication Date (Web): 10 Apr 2017 Downloaded from http://pubs.acs.org on April 12, 2017

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ACS Applied Materials & Interfaces

Enhancing Vascularization through the Controlled Release of Platelet-Derived Growth Factor -BB Silvia Minardi †+, Laura Pandolfi†§+, Francesca Taraballi†, Xin Wang†, Enrica De Rosa†, Zachary D. Mills†, Xuewu Liu‡, Mauro Ferrari‡ and Ennio Tasciotti† * Φ

AUTHOR ADDRESS † Center for Biomimetic Medicine, Houston Methodist Research Institute, 6670 Bertner Ave. Houston, TX 77030 (USA). § College of Materials Science and Engineering, University of Chinese Academy of Science, 19A Yuquanlu, Beijing, China. ‡ Department of Nanomedicine, Houston Methodist Research Institute, 6670 Bertner Ave. Houston, TX 77030 (USA). ΦDepartment of Orthopedics, Houston Methodist Hospital, 6565 Fannin St, Houston, TX 77030, (USA). KEYWORDS. porous silicon; PLGA; drug delivery; PDGF-BB; tissue engineering. ABSTRACT: Using delivery systems to control the in vivo release of growth factors (GFs) for tissue engineering applications is extremely desirable as the clinical use of GFs is limited by their fast in vivo turnover. Hence, the development of effective platforms, able to finely control the release of GFs in vivo, remains a challenge. Herein, we investigated the ability of multiscale composite microspheres, based on a nanostructured silicon multistage vector (MSV) core and a poly(DLlactide-co-glycolide) acid (PLGA) outer shell (PLGA-MSV), to release functional platelet-derived growth factor-BB (PDGFBB), to induce in vivo localized neo-vascularization. The in vitro release of PDGF-BB was assessed by enzyme-linked immunosorbent assay over 2 weeks, and showed a sustained, zero-order release kinetic. The ability to promote in vivo localized neovascularization was investigated in a subcutaneous injection model in BALB/c mice, and followed by intravital microscopy up to 2 weeks. Fully functional newly formed vessels were found within the area where PLGA-MSVs were localized, and found to cover 3.0±0.9% and 19±5.1% at 7 and 14 days, respectively, showing a 6-fold increase in one week. The distribution of CD31+ and α-SMA+ cells was detected by immunofluorescence on harvested tissues. CD31 resulted significantly more expressed (4-fold increase), compared to the untreated control. Finally, the level of up-regulation of angiogenesis-associated genes (Vegfa, Vwf, and Col3a1) was assessed by q-PCR, resulting in a significantly higher expression where PLGA-MSVs were localized (Vegfa: 2.32±0.50 at 7 days and 4.37±0.75 at 14 days; Vwf: 4.13±0.82 and 7.74±0.91; Col3a1: 5.43±0.37 and 6.66±0.89). Altogether, our data supported the conclusion that the localized delivery of PDGF-BB from PLGA-MSVs induced the localized de-novo formation of fully functional vessels, in vivo. With this study, we demonstrated that PLGA-MSV holds promise for accomplishing the controlled localized in vivo release of GFs, for the design of innovative tissue engineering strategies.

1. INTRODUCTION.

The ability of growth factors (GFs) to orchestrate cellular activities such as proliferation, 1 migration,2 and differentiation,3, 4 makes them valuable tools in promoting tissue repair.5-7 The controlled release of GFs by delivery systems has proved a valuable strategy in different fields of tissue engineering.8, 9 The ideal delivery platform should be fully customizable to suit various applications and accommodate different payloads. Moreover, it should allow for the storage of large amounts of payload.10 Several carriers have shown to prolong and sustain GFs release,11 reducing side effects and toxicity.12-14 However, major limitations still need to be addressed: (i) high costs; (ii) loss of GFs bioactivity upon encapsulation; (iii) non-specific distribution of GFs after administration.11 The loss of bioactivity upon encapsulation not only affects the desired therapeutic outcome, but also requires the use of higher doses of bioactive molecules, which has been associated with disruptive side effects.15 Although the amount of GF to be used in clinical practice is still con-

troversial, controlling their localization after administration is key, as it would enable to reduce the necessary dosage, significantly favoring their clinical translation, while reducing their cost.16 In order to overcome the present limitations, various nanostructured delivery systems have been proposed.8, 17 Among developed platforms, composite microparticles, based on biodegradable polymers and inorganic silicon or silica,10, 18 have been demonstrated to be particularly advantageous, allowing for a greater variety of formulations, which can be designed ad hoc to suit potentially any tissue engineering application.19, 20 In the last decades, poly lactic-co-glycolic acid (PLGA) was established as the polymer of choice for the fabrication of delivery systems due to its versatility and low toxicity,21 and has been proved to facilitate the delivery of multiple payloads.22 Porous inorganic silicon or silica microparticles have been successfully utilized in different fields,23-25 allowing for increased loading of a variety of payloads, compared to other materials.26, 27 In particular, nanostructured silicon

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multistage vectors (MSVs) have been extensively studied for multiple applications ranging from cancer,25, 28-30 to tissue engineering,11, 20 due to their low toxicity, reproducibility, and tunability (e.g. shape, size, pore size, overall porosity and degradation).18 Furthermore, MSVs’ surface can be chemically modified to enhance protein loading efficiency.9, 31 MSVs encapsulated in a PLGA shell exhibited greater control over the release kinetics of proteins.10, 11 and small molecules,32 by ameliorating their burst release in vivo.19 Among the regenerative processes that could be trigged by GFs, neovascularization is crucial to tissue engineering. The formation of new vasculature is fundamental to powering the regenerative niche that eventually forms after injury.33 The new formation of a mature vascular network is directed by a series of bioactive molecules regulating the proliferation of endothelial cells that delineate blood vessels.34 Some of the most highly characterized molecules involved in the regulation of angiogenesis include vascular endothelial growth factor (VEGF),35 basic fibroblast growth factor (b-FGF),36 and platelet-derived growth factor (PDGF).37 PDGF has several isoforms (AA, AB, BB, CC, and DD),38-40 which signal through two distinct receptors.41, 42 PDGF-BB is the only isoform able to bind to all known receptors.43 PDGF-BB is particularly attractive for tissue engineering, as it not only actively participates in angiogenesis, but also contributes to osteogenesis and mesengenesis.8, 18 However, previous studies demonstrated that angiogenic GFs have no significant therapeutic effects if administered through bolus injection, due to their short half-life.44, 45 Therefore, delivery systems may represent a valuable strategy to overcome this limitation.8 Herein, we investigated the ability of PLGA-MSV composite microspheres to preserve the functionality of PDGFBB, while avoiding its burst release, to promote localized neo-vascularization, in a subcutaneous model in BALB/c mice. Endpoints included the assessment of PDGF-BB in vitro controlled release, in vitro test of PLGA-MSVs with human umbilical vein endothelial cells (HUVECs), intravital microscopy analysis, immunofluorescence, histology and q-PCR to evaluate and quantify the extent of localized newly formed vessels in vivo, upon PDGF-BB release from PLGA-MSVs.

2. MATERIALS AND METHODS 2.1. Fabrication and characterization of MSVs Discoidal MSVs particles with diameters of 1 μm were fabricated by photolithography and electrochemical porosification of patterned silicon wafers, as extensively described elsewhere.18 MSVs were then oxidized and their surface modified with (3-aminopropyl)-triethoxysilane (APTES) (Sigma-Aldrich), as reported previously.46 Modified MSVs were coated with 3 nm of Pt/Pb, and their nanostructure evaluated by imaging at a voltage of 7 kV, using a scanning electron microscopy (SEM) (FEI Quanta 400 SEM FEG).

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2.2. Loading of PDGF-BB into MSVs The loading of PDGF-BB in PLGA-MSVs was obtained by incubating MSVs particles, for 2 hours at 37°C, under mild agitation. In detail, 2x108 APTES-modified MSVs were suspended in 500 μL of solution of PDGF-BB in phosphate buffer saline (PBS) (30 µg/mL) (Thermo Fisher Scientific). The loaded particles were then recovered through centrifugation at 4500 rpm for 10 min. The supernatant was used to determine the amount of PDGF-BB loaded in the MSVs particles by Enzyme-Linked Immunosorbent Assay (ELISA) (R&D Systems Inc.), as extensively described elsewhere.10, 11, 47, 48 The PDGF-BB-loaded MSVs were lyophilized overnight and then encapsulated in the 10% 50:50 PLGA solution, as described in paragraph 2.3. 2.3. Fabrication and characterization of PLGA-MSVs 2×108 APTES modified MSVs or PDGF-BB-loaded MSVs were encapsulated in PLGA (LACTEL) via a modified double-emulsion method, as previously described.11 Briefly, 50:50 PLGA was dissolved in dichloromethane (DCM) (Sigma-Aldrich) at a concentration of 10% w/v. MSVs were mixed in 1 mL of 10% w/v PLGA in DCM, then dropped into 3 mL of a solution of poly(vinyl alcohol) (PVA) (Sigma-Aldrich) 2.5% w/v, and subsequently emulsified at 3500 rpm for 10 minutes, with a homogenizer (IKA Eurostar). The resulting emulsion was dropped into 40 mL of PVA 1% w/v and stirred overnight to allow DCM evaporation. Particles were washed three times with distilled water, by sequential centrifugation. The microspheres were finally recovered and freeze-dried and stored under vacuum. To fabricate the fluorescently labelled PLGA-MSVs utilized in the intravital miccroscopy experiment, fluorescein isothiocyanate (FITC) (SigmaAldrich) or Tetramethylrhodamine (TRITC) (Thermo Fisher Scientific) was added to the PLGA solution in DCM at a concentration of 1 mg/mL.9 PLGA-MSVs were imaged by scanning electron microscopy (SEM) and their size distribution was measured with the software ImageJ (NIH Image). 2.4. PDGF-BB in vitro release from PLGA-MSVs Lyophilized PDGF-BB-loaded PLGA-MSVs (PLGAMSV/PDGF-BB) were resuspended in PBS (1.5 mL), and subsequently divided into three aliquots (0.5 mL each). The release study was performed at 37ºC, under mild agitation (200 rpm). Samples were collected, at established time intervals, for 2 weeks. Each sample was centrifuged (4000 rpm; 10 min), and 10% of the supernatant (0.05 mL) was collected and replaced with an equal volume of PBS. The amount PDGF-BB released at each time point was determined by ELISA (R&D Systems Inc.). 2.5. HUVECs Culture HUVECs cultures were established following the manufacturer's instructions (Lonza). HUVECs were seeded (2000 cells/cm2) in 8-well chamber slides and cultured in endothelial growth media (Lonza). After 24 h from seeding, to allow cells to properly adhere to the chambers, TRITC-labeled PLGA-MSV (PLGA-MSV-TRITC) were added at a concentration of 10 μg/mL. TRITC-labelled


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PLGA-MSVs were synthesized as previously described.9 At 72 h, cells were washed three times with PBS and fixed with 4% paraformaldehyde (Sigma-Aldrich) for 20 minutes, at room temperature. Cells were finally washed three times with PBS prior to labeling with DAPI (nuclei) and phalloidin-488 (actin) (Life Technology), for fluorescence imaging by confocal laser microscopy. 2.6. Subcutaneous Injections of PLGA-MSV/PDGF-BB A total of 26 BALB/c mice (female, 8−12 weeks old) were housed in the animal facility at The Houston Methodist Research Institute (Houston, TX, USA) for two days before starting the experiment. The Comparative Medicine Program (CMP) of the Houston Methodist Research Institute, in its role as the Institutional Animal Care and Use Committee (IACUC), approved the animal protocol (protocol: AUP-0115-0002) and ensured a current Animal Welfare Assurance (#A4555-01) with the Office of Laboratory Animal Welfare (OLAW). Mice were anesthetized with 2% isoflurane prior to injection, to allow for the comfortable shaving and sterilization of the skin, at the site of injection. Of the 26 mice, 18 were used for imaging by intravital microscopy, at 7 and 14 days (n = 9). At each time point, after imaging, specimens were collected and analyzed by qPCR (n=6, total analyzed). The remaining 6 mice were used for immunofluorescence and histological evaluation, at 7 and 14 days (n = 9). Each mouse received three dorsal subcutaneous injections of 50 μL of a PLGAMSV/PDGF-BB suspension in PBS, and one injection of 50 μL of empty PLGA-MSV in PBS, as a control. We maintained a consistent dose (150 ng) of PDGF-BB delivered by PLGA-MSV/PDGF-BB for each injection. At the experimental time points, animals were euthanized by CO2 inhalation and subsequent cervical dislocation. 2.7. Intravital Microscopy Intravital microscopy was performed according to the animal protocol approved by the IACUC of Houston Methodist Research Institute (protocol: AUP-0614-0039). The surgical procedure was performed under aseptic conditions. Pre-operative analgesic (Caprofen/Bupronex) was administered before surgical operation. The animals were anesthetized with 2% isoflurane, and their respiratory rate monitored throughout the procedure. Under deep anesthesia, the site of injection of FITClabelled PLGA-MSVs or PLGA-MSV/PDGF-BB was exposed through the surgical dissection of the skin, while an intact blood supply was maintained. Mice were then transferred to the observation stage under the microscope, and Texas-Red labeled 70 kDa dextran (Thermo Fisher Scientific) was administered via retro-orbital injection to highlight blood vessels and assess localized neovasculature formation. Images and videos were acquired after injection using an upright A1R laser scanning confocal microscope (Nikon) equipped with a resonance scanner, motorized and heated stage. All settings, including laser power, gain, offset, and pinhole diameter were maintained throughout each acquisition. At 14 days, animals were injected with red blood cells stained with Vybrant® DiD (Thermo Fisher Scientific) to evaluate the functional-

ity of the newly formed vessels. Animals were sacrificed after imaging and tissues collected for further analysis (n=12 for the mice used as native controls; n=9 for the other experimental groups). Images were analyzed by NIS-Elements software (Nikon). At least 3 different areas per mouse have been analyzed to quantify new vessel formation (area fraction) and networking (nodes number) and vessel diameter. Per each area, 5 images were analyzed and the data averaged. To determine the mean diameter of the vessels, at least 20 measurements were acquired. The red blood cells flow rate at 14 days was calculated through an automated tracking tool of the NIS Elements software (Nikon). 2.8. Immunofluorescence For the staining of in vitro samples with phalloidin and DAPI, we followed standard protocols, suggested by the manufacturer (Life Technology). Prior to staining, the samples were washed in PBS three times, and fixed with 4% paraformaldehyde (PFA) (Sigma Aldrich) for 20 minutes at room temperature. Cells were permeabilized by incubation with a solution 0.1% Triton-X100 (SigmaAldrich) in PBS. For the staining of the in vivo samples (n=9, per each experimental group), at 7 and 14 days, the tissue surrounding the site of each injection was collected and embedded in optimum cutting temperature (OCT) mounting media (Tissue Tek). Frozen tissues were cut 10 μm thick, through a cryostat (Thermo Fisher Scientific). Sections were washed twice in xylene and rehydrated sequentially with decreasing ethanol concentrations (100%, 95%, 90%, 80%, and 70%) and distilled water. Tissue slides were stained for alpha-smooth muscle Actin (α-SMA) (Abcam), CD31 (LSBio) and DAPI (Sigma-Aldrich) according to manufacturers’ protocols. Samples were imaged with a confocal laser microscope (A1 Nikon Confocal Microscope) and the images were analyzed through the NISElements software (Nikon). 2.9. Histological Evaluation The tissue slides (n=9, per each experimental group) were also stained with Masson’s trichrome staining (Abcam), according to the manufacturer’s protocol. Stained slices were mounted with Cytoseal XYL (Thermo Scientific) mounting medium, and analyzed by histological microscope (ECLIPSE Ci-E, Nikon). 2.10. Gene Expression Analysis At 7 and 14 days, the tissue surrounding the site of injection of PLGA-MSVs and PLGA-MSV/PDGF-BB was harvested and lysed in 500 µL of Trizol reagent (Invitrogen) (n = 6, per each experimental group). mRNA was extracted according to manufacturer’s protocol. DNAse (SigmaAldrich) treatment followed. RNA concentration and purity were measured by a NanoDrop ND1000 spectrophotometer (NanoDrop Technologies). cDNA was reverse transcribed from 1 µg of total RNA with iScript retrotranscription kit (Bio-Rad Laboratories). Amplifications were carried out using TaqMan® Fast Advanced Master Mix on a StepOne Plus real-time PCR system (Applied Biosys-



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tems). The following probes (Applied Biosystems) were used to evaluate the expression of in situ neovascularization: (i) vascular endothelial growth factor A, Vegfa (Mm01281449_m1); (ii) Von Willebrand factor homolog, Vwf (Mm00550376_m1); (iii) collagen type III alpha 1, Col3a1 (Mm01254476_m1). The expression of each gene was first normalized to the housekeeping gene Gapdh (glyceraldehyde-3-phosphate dehydrogenase, Mm99999915_g1). Relative fold changes in gene expression were finally normalized to those obtained from native tissue (mice injected with PBS, CTRL). The results were reported as mean ± standard deviation. 2.11. Statistical Analysis Statistical analysis was performed via Prism GraphPad software. A two-way ANOVA was used. In all cases * was used for p < 0.05, ** for p < 0.01, *** for p < 0.001, and **** for p < 0.0001. For gene expression analysis, one-way analysis of variance for multiple comparisons by the Student–Newman–Keuls multiple comparison test was used, as previously reported.49 Experiments were performed at least in triplicate. Data are presented as mean ± standard deviation.

3. RESULTS AND DISCUSSION 3.1. Fabrication and Characterization of PLGAMSV/PDGF-BB Controlling the size and shape of a delivery platform is fundamental to achieve an advantageous loading efficiency and obtain the required release kinetics. Herein, we proposed a fully tunable platform for the in vivo release of PDGF-BB (PLGA-MSV/PDGF-BB). PLGA-MSV/PDGF-BB were obtained through a modified double-emulsion method, as extensively described elsewhere,10 and area based on an inorganic core (MSV particle) (Fig 1B) loaded with PDGF-BB, and an organic polymer shell (PLGA). Embedding MSVs in a polymeric shell has been demonstrated to delay the release of the payload, helping to reduce or eliminate the initial burst release.46 In a recently published study, we showed how both components of this delivery system can be tuned and adjusted, depending on the final application.19 The production of MSVs through lithography is precise, scalable, and reproducible, fabricating MSVs with a uniform size, porosity and full control over their geometry.18, 25 In this study, we used discoidal MSVs with a diameter of 1 μm and a total 51% of porosity (Fig 1B). For the PLGA outer layer, a copolymer ratio of 50:50 and a viscosity of 0.55-0.75 dL·g-1 was chosen, in order to achieve the desired release kinetics of PDGF-BB within 2 weeks, according to our previous studies.19 The morphology of the resulting PLGA-MSVs was characterized by SEM. The micrograph in Fig 1A shows microspheres of uniform shape and size. The mean size distribution of the microspheres was 8.2 ± 2.1 μm (Fig 1C).

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To increase the loading efficiency of PDGF-BB in MSV particles, their surface was oxidized and then APTES modified. 50 In this specific study, we achieved an average loading efficiency of 76.7 ± 3.2%. The loading is orchestrated by capillary forces and electrostatic interactions that act during the immersion of MSVs into the loading solution.51 The modified surface of the pore walls augmented the interactions between the payload and the pore walls,52 favoring the docking of the payload and increasing the amount of loaded molecules within the carrier. 3.2. In vitro release of PDGF-BB The release of PDGF-BB was performed in physiologicallike conditions, for 2 weeks. The mass of PDGF-BB released from the carrier was assessed by ELISA, using the collected supernatants at different time points during the experiment; the cumulative release profile is shown in Fig 2A. After two weeks, the release of PDGF-BB from the composite microspheres reached approximately 100% of the payload. As it can be seen from the release profile, the delivery platform minimized the initial burst release typical of most polymeric delivery systems.53 In particular, the zero-order release kinetic was mainly accomplished in the first 7 days of release, with a linear regression curve featuring a R2 of 0.992, compared to a R2 of 0.951 for the whole data set (S1 Fig). The platform accomplished a release of PDGF-BB of approximately 43.9 ± 8.1 pg·day-1 (Fig 2B). The sustained and controlled release of the payload has been shown to be crucial to ensure proper therapeutic outcomes and avoid undesired side effects.54 The release kinetics of PDGF-BB, in fact, are associated with the degradation of PLGA-MSV, which have been shown to exhibit a more sustained and slow degradation process over time.9, 10 3.3. In vitro testing of PLGA-MSVs with HUVECs We tested PLGA-MSV in vitro, with HUVECs (Fig 3), and we found that when incubated with PLGA-MSV, the cells displayed the typical shape of endothelial cells, forming a monolayer (Fig 3A). Also, the actin of HUVECs’ cytoskeleton did not present abnormalities. At higher magnifications, it was possible to observe that, at 72 h, cells did not internalize PLGA-MSV. PDGF-BB, as others GFs and cytokines, function by binding to surface cell receptors, activating a transmembrane signal transduction.55 Towards this end, the ligand (GF) has to be released in the extracellular space of tissues. 56 Nano-sized particles are easily internalized upon contact with cells,57 and MSVs have been shown to be internalized by cells within a few hours both in vitro and in vivo.25, 58 In this particular study we assure that PLGA-MSV neither altered the targeting cells nor have been internalized by them. In a previous study, we investigated the ability of macrophages (both J774 and bone marrow-derived macrophages) to internalize PLGAMSVs in vitro, to simulate the in vivo response of phagocytic cells. Firstly, we demonstrated that the viability of macrophages was not compromised by increasing concentrations of PLGA-MSVs, and that their size prevented the uptake.9 Secondly, we assessed the biocompatibility of


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PLGA-MSVs in vivo, by quantifying the amount of silicon in BALB/c mice’s organs, after subcutaneous injection of PLGA-MSVs. We found that most PLGA-MSVs were retained within the site of injection up to 2 weeks. This finding supported our hypothesis that PLGA-MSVs could remain localized for 2 weeks in vivo, accomplishing a localized released of their payload.9 Coating MVSs with a polymeric outer shell revealed particularly advantageous to prevent internalization, as we previously demonstrated.11 3.4. Evaluation of in vivo neovascularization Neovascularization is highly desired in many applications of tissue regeneration.59, 60 However, the in vivo release of growth factors are associated with detrimental side effects (including cancer), mainly due to the uncontrolled release of the factors, and currently limits their clinical use.15, 61 One of the goals of this study was to determine if PLGAMSVs could serve as an efficient delivery system for the preservation and controlled release of PDGF-BB in vivo. We had previously demonstrated that PLGA-MSVs were able to release a controlled daily dose of a reporter protein (FITC-BSA) over 2 weeks, while avoiding the initial burst release, in a mouse subcutaneous injection model.9 We had also showed that the reporter protein was released only in proximity of the site of injection of the PLGA-MSVs. This further supported the hypothesis that PLGA-MSVs could not only control the release of proteins in time, but also control their localization in vivo, accomplishing a spatiotemporally controlled release. In the present study, we wanted to assess the possibility to accomplish a temporal and spatial control over the release of a bioactive molecule through the PLGA-MSV, for a controlled and localized angiogenesis, in a murine model, leveraging on our previous findings. FITC-labeled PLGAMSVs and PLGA-MSV/PDGF-BB were injected subcutaneously in mice. New vessel formation was evaluated through intravital microscopy. Fig 4 shows the images and characterization of the vessels formed within the site of injection of PLGA-MSVs and PLGA-MSV/PDGF-BB. In the control group (PLGA-MSV), no significant evidence of new vessel ingrowth was found. In fact, the only vessels visible were those normally present in the tissue of the mice, but no branching was found in the area where the microspheres were localized, neither at 7 (Figs 4A, B) nor at 14 days (Figs 4C, D). On the contrary, in the PLGAMSV/PDGF-BB group, newly formed vessels were identified and found to cover 3.0 ± 0.9% of the area where the microspheres were localized at 7 days (Figs 4E, F), and 19 ± 5.1% at 14 days (Figs 4G, H), showing of a 6-fold increase between the two time points (Fig 4I). The newly formed vessels appeared to sprout from the adjacent vessels and to form an intricate vascular network within the area where PLGA-MSV/PDGF-BB were injected, as indicated by white arrows in Fig 4. The diameter of the newly formed vessels was measured and compared to that of native tissue (Fig 4L). Native vessels were found to have a mean diameter of 33.6 ± 9.5 μm while the newly formed ones within the PLGA-MSV/PDGF-BB were 27.5 ± 8.6 μm at 7 days and 21.4 ± 0.7 μm at 14 days. Finally, the extent

of branching of the newly formed capillary network was estimated by counting the mean number of nodes per group, compared to control native tissue. It was found that the native capillary network had an average number of 3.0 ± 1.6 nodes per mm2, while the vessels formed in the area where PLGA-MSV/PDGF-BB were localized had an average of 16.0 ± 5.5 nodes per mm2. The significantly higher degree of branching of the vessels formed in response to the released PDGF-BB could be attributed to the complex network of interconnected pores formed by the microspheres. To assess the functionality of the vessels formed in response to PLGA-MSV/PDGF-BB, we injected fluorescently labelled red blood cells, and evaluated their flow in the newly formed vessels. We observed that, within a few seconds, the injected red blood cells reached the vessels, demonstrating that the newly formed vascular network was fully functional (as shown in Supplementary Video S1 and Video S2). Consistently with our data previously published,9 PLGA-MSV were retained within the site of injection even after 2 weeks, as shown in the supplementary information (S2 Fig). No significant amount of particles was identified in the surrounding areas. The flow rate of the red blood cells was measured as 257.7 ± 161.2 μm/sec for the tissue of mice injected with PBS (CTRL native) and 418.0 ± 176.0 μm/sec in the PLGA-MSV/PDGF-BB group (S3 Fig). The high standard deviations found also in the CTRL native group reflect the physiological variability of diameter and blood flow rate of vascular networks. Furthermore, the discrepancy between CTRL native and the PLGA-MSV/PDGF-BB group to the smaller diameter of the newly formed vessels, as reported in Fig 4. To confirm that the newly formed vessels, found in correspondence of PLGA-MSV/PDGF-BB, were functional, we also performed immunofluorescence staining of CD31 and α-SMA markers, to identify endothelial cells and smooth muscle cells, respectively, which are normally both present in vessels.62, 63 Data are reported in Fig 5. The group treated with PLGA-MSV/PDGF-BB recruited a higher overall amount of CD31+ cells, compared to PLGA-MSV (Figs 5A, B, and S4 Fig in supplementary information). Furthermore, by quantifying the amount of CD31 in the control group and PLGA-MSV/PDGF-BB, we found that CD31 was significantly more expressed (4-fold increase) in the group treated with PDGF-BB, and co-localized with the vessels (Fig 5C). To further assess this outcome, the area of tissue in which PLGA-MSV were localized was collected after 14 days and sliced both in cross (Fig 5D) and longitudinal sections (Fig 5E). It was possible to identify several vessels positive for both CD31 and α-SMA, and that α-SMA+ cells were organized perpendicularly to the vessel, wrapped around CD31+ cells, which, on the contrary, were organized longitudinally, confirming they were fully organized vessels (S5 Fig.). Furthermore, the staining of CD31 in the longitudinal sections displayed an interconnected and extended vascular network. The fluorescent signal associated with α-SMA, was significantly higher in the PLGA-MSV/PDGF-BB group (~5-fold increase), compared to PLGA-MSV group (Fig 5F). The overall area covered by of the CD31+ and α-SMA+ cells in the PLGA-



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MSV/PDGF-BB group was quantified, to determine their density in the tissue, and it was found to be 25% (±3.0) and 9% (± 0.8), respectfully. On the contrary, in the PLGA-MSV group (CTRL), the density of CD31+ and αSMA+ cells was 8% (± 0.1) and 2% (± 0.0). Since PDGF-BB has been found to contribute to the regeneration of multiple tissues (e.g. bone), it has become greatly attractive in a multiplicity of tissue engineering approaches.64, 65 The tissue formed within the site of injection of PLGAMSVs releasing PDGF-BB was evaluated by histological analysis through Masson’s trichrome staining, to assess the impact of the delivery systems on the surrounding tissue, at 7 and 14 days (Fig 6). We noticed a considerably higher amount of cells being recruited in the mice treated with PLGA-MSV/PDGF-BB, compared to the control group (PLGA-MSV). The PLGA-MSV group did not show any sign of neovascularization, neither at 7 (Figs 6A, B) nor at 14 days (Figs 6C, D). Also, it can be observed that over time, the number of cells within the tissue decreased, and that new fibrous extracellular matrix (stained in blue) was deposited (Figs 6C, D). At 7 days, a significant difference between the PLGA-MSV and the PLGA-MSV/PDGF-BB groups was already visible (Figs 6E, F). Histological evaluation confirmed the presence of a wide interconnected newly formed vascular network at 7 days, which grew further over time. In Figs 6G and H, yellow arrows indicate the vessels with a fully formed lumen. The histological sample showed in Fig 6H was also sliced longitudinally, to visualize the whole vascular network; images are reported in the supplementary information (S6 Fig). Finally, to quantify the up-regulation of angiogenesisassociated marker genes (Vegfa, Vwf, and Col3a1), the tissue surrounding the site of injection of the microspheres was collected and analyzed by q-PCR, at 7 and 14 days. The results were normalized to that obtained for the

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control group (mice injected with PBS - CTRL), and are reported in Fig 7. Injection of empty PLGA-MSVs did not affect the expression of any of the selected genes. The only notable relative-fold increase was observed in the PLGA-MSV/PDGF-BB group (Vegfa: 2.32 ± 0.50 at 7 days and 4.37 ± 0.75 at 14 days; Vwf: 4.13 ± 0.82 and 7.74 ± 0.91; Col3a1: 5.43 ± 0.37 and 6.66 ± 0.89), demonstrating that PLGA-MSV/PDGF-BB significantly induced localized new vessel formation, consistently to what observed through intravital microscopy, immunofluorescence and histology.

4. CONCLUSIONS The scope of this study was to assess the use of PLGAMSVs as a delivery platform for the spatiotemporally controlled release of PDGF-BB, promoting the localized formation of functional vessels upon subcutaneous injection in mice. Our main findings were that: (i) through optimized loading conditions, PDGF-BB was encapsulated in the carrier at high efficiency and released in a controlled fashion, characterized by a significant reduction of the initial burst release; (ii) in vivo, PLGA-MSVs remained localized within the initial site of injection over 2 weeks; (iii) PLGA-MSVs released functional PDGF-BB, as a newly formed functional vascular network was found in the PLGA-MSV/PDGF-BB group, and found to cover 3.0 ± 0.9% and 19 ± 5.1% at 7 and 14 days, respectively, showing a 6-fold increase between the two time points. These findings correlated with the results obtained by immunofluorescence, which showed a 4-fold increase of CD31+ cells in the treated group, and with the data obtained by q-PCR. Altogether, this data proved PLGA-MSV as an efficient platform for the in vivo controlled release of functional PDGF-BB. This study provides new evidence in support of the future use of PLGA-MSVs in the loading and release of GFs for tissue engineering applications.

Fig 1. PLGA-MSV. The overall morphology of PLGA-MSV microparticles was assessed through SEM imaging. (A) SEM micrograph of PLGA-MSV microspheres illustrating their typical shape and size uniformity (6.7 ± 1.3 µm). (B) SEM images of individual discoidal MSV that shows their size (1 μm) and porosity (51%). (C) Size distribution of PLGA-MSV.


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Fig 2. PDGF-BB in vitro release. (A) Cumulative release profile of PDGF-BB from PLGA-MSV showing an almost zeroorder release, interpolated by a linear regression curve and corresponding mean square error. (B) Daily release of PDGFBB from PLGA-MSV (43.9 ± 2.8 pg).

Fig 3. In vitro testing of PLGA-MSV with HUVECs. Confocal laser microscopy images 72 h after HUVECs were incubated with PLGA-MSV-TRITC microspheres (red) at different magnifications, 20X (A), 40X (B), and 60X (C).



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Fig 4. Intravital microscopy. Intravital microscopy images at 7 and 14 days after PLGA-MSV (A-D) and PLGAMSV/PDGF-BB (E-H) were injected subcutaneously in mice. PLGA-MSV are in green, while vessels are in red. Overall amounts of newly formed vessels in the site of injection (I). Diameter and number of nods of the newly formed vessels (L and M, respectively). Values are reported as mean ± standard deviation. A value of p < 0.05 was considered statistically significant: ** p < 0.01; ***p < 0.001; ****p < 0.0001.


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Fig 5. Immunohistochemistry. Confocal laser microscopy images evaluating in vivo neovascularization facilitated by the controlled release of PGDF-BB at 14 days. (A, B) The CTRL and treated groups stained for phalloidin (green) and CD31 (red) to evaluate overall vessel organization and maturation. (C) Quantification of CD31. (D) Cross and longitudinal sections (E) of the subcutaneous pouch have been further stained with CD31 (red) and α-SMA (green) to better visualize the vascular network formed and to confirm the presence of smooth muscle cells within this network. (F) Quantification of αSMA, compared to the control. Values are reported as mean ± standard deviation. A value of p < 0.05 was considered statistically significant: ** p < 0.01; ***p < 0.001; ****p < 0.0001.



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Fig 6. Histological evaluation. Histological evaluation by Masson’s trichrome staining at 7 (A-D) and 14 days (E-H) of cross sections of the in vivo vessel formation guided by PDGF-BB loaded PLGA-MSV, injected subcutaneously in a mouse model. Control mice were injected with PBS and compared to mice injected with PLGA-MSV/PDGF-BB. In the control groups, no sign of new vessel formation was observed. On the contrary, the controlled release of PDGF-BB over 14 days guided what appeared to be a mature network of vessels (yellow arrows).

Fig 7. Quantitative PCR. q-PCR analysis for the expression of vasculature genes (Vegfa, Vwf, and Col3a1) for CTRL, PLGA-MSV, and PLGA-MSV/PDGF-BB at 7 and 14 days. Data are represented as relative fold expression (normalized to CTRL). Values are mean ± SD. Asterisks depict highly significant differences (* p < 0.05, ** p < 0.01).


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ASSOCIATED CONTENT (Word Style “TE_Supporting_Information”). Supporting Information. S1 Fig. Interpolated trendline of the cumulative release curve. / S2 Fig. Intravital microscopy images at 14 days of PLGA-MSV/PDGF-BB. / S3 Fig. Red blood cells flow rate in newly formed vessels. / S4 Fig. Confocal laser microscopy of native mouse skin. S5 Fig. Analysis of a fully formed α-SMA+CD31+ vessel. / S6 Fig. Masson’s trichrome staining at 14 days. / Video S1. Intravital microscopy. Red blood cells circulating in the whole vascular network, that formed where the PLGA-MSV/PDGF-BB were localized. / Video S2. Intravital microscopy. Higher magnification of red blood cells circulating in the entirety of the newly formed vascular network in the PLGA-MSV/PDGF-BB group. This material is available free of charge via the Internet at http://pubs.acs.org.” AUTHOR INFORMATION

Corresponding Author * E. Tasciotti Department of Regenerative medicine, Houston Methodist Research Institute, 6670 Bertner Ave, Houston, TX 77030 (USA). E-mail: [email protected] Addresses ^ S. Minardi, Current address: Northwestern University, Feinberg School of Medicine, Department of Medicine, 320 E Superior St, Searle 10-450 Chicago, Illinois 60611; [email protected]

Author Contributions The manuscript was written through contributions of all authors. / SM, FT conceived the idea of this manuscript, and designed the experimental plan, with the help of LP. SM and ZDM fabricated the PLGA-MSV/PDGF-BB and performed the release study. LP and FT characterized PLGA-MSV. LP performed injections in mice. SM performed light microscopy. EDR performed intravital microscopy; SM analyzed the data. XW performed q-PCR and data analysis. XL provided MSV particles. MF and ET supervised the study and provided funds. / All authors have given approval to the final version of the manuscript. / +These authors contributed equally.

Funding Sources The project was funded by the Brown Foundation (Project ID, 18130011) and by the Cullen Trust for Health Care Foundation (Project ID, 18130014). The work was supported by funds from the Houston Methodist Research Institute. Partial funds were acquired from the Ernest Cockrell Jr. Presidential Distinguished Chair (M.F.).

ACKNOWLEDGMENT We thank Dr. J. Gu of the HMRI Microscopy-SEM/AFM core, and Dr. Kemi Cui of the HMRI ACTM core. We thank Dr. Fernando Cabrera for his help with the animal study and the animal protocol, and Nupur Basu for help with cell culture.

ABBREVIATIONS

MSV, nanostructured silicon particles; PLGA, poly(dl-lactideco-glycolide) acid; PLGA-MSV, PLGA-porous silicon particles composite microspheres; PDGF-BB, Platelet-Derived Growth Factor-BB; PLGA-MSV/PDGF-BB, PLGA-porous silicon particles composite microspheres, loaded with PDGF-BB.

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15

Enhancing Vascularization through the Controlled Release of Platelet

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