Polyplex transfection from intracerebroventricular delivery is not significantly affected by traumatic brain injury

Abstract

Traumatic brain injury (TBI) is largely non-preventable and often kills or permanently disables its victims. Because current treatments for TBI merely ameliorate secondary effects of the initial injury like swelling and hemorrhaging, strategies for the induction of neuronal regeneration are desperately needed. Recent discoveries regarding the TBI-responsive migratory behavior and differentiation potential of neural progenitor cells (NPCs) found in the subventricular zone (SVZ) have prompted strategies targeting gene therapies to these cells to enhance neurogenesis after TBI. We have previously shown that plasmid polyplexes can non-virally transfect SVZ NPCs when directly injected in the lateral ventricles of uninjured mice. We describe the first reported intracerebroventricular transfections mediated by polymeric gene carriers in a murine TBI model and investigate the anatomical parameters that dictate transfection through this route of administration. Using both luciferase and GFP plasmid transfections, we show that the time delay between injury and polyplex injection directly impacts the magnitude of transfection efficiency, but that overall trends in the location of transfection are not affected by injury. Confocal microscopy of quantum dot-labeled plasmid uptake in vivo reveals association between our polymers and negativel G2 chondroitin sulfate proteoglycans of the SVZ extracellular matrix. We further validate that glycosaminoglycans but not sulfate groups are required for polyplex uptake and transfection in vitro. These studies demonstrate that non-viral gene delivery is impacted by proteoglycan interactions and suggest the need for improved polyplex targeting materials that penetrate brain extracellular matrix to increase transfection efficiency in vivo.

GRAPHICAL ABSTRACT

1. Introduction

Approximately 1.7 million people suffer a traumatic brain injury (TBI) each year in the United States alone, injuries that can result in death or permanent disability while costing the healthcare system over $50 billion annually. [1,2] While focal TBI is initiated as an acute blunt-force trauma to the exterior of the skull that causes immediate cortical zone cell death, larger ischemic or swollen regions then develop throughout the cortex that produce permanent scarring and neuron loss.[3] The current standard of care for TBI seeks to curtail these secondary damages by limiting bleeding and inflammation but requires prompt implementation and does not ultimately restore the function of the neural tissue lost to injury.[4] This is in part because the injured central nervous system (CNS) has limited endogenous neurogenic capacity and the injury environment negatively impacts neural precursor migration, differentiation and functional integration.[5] Thus, therapeutic strategies for the regeneration and integration of neurons at the site of injury could greatly impact patient recovery after TBI.

While stem cell transplantation therapies for tissue regeneration in the CNS have rapidly progressed into clinical study,[6] gene therapies that manipulate endogenous neural progenitor cells (NPCs) with transcription or growth factors offer a promising alternative.[7–9] Compared to cultured cell therapies used as transplants, “direct reprogramming” approaches are more cost effective, less toxic, and promise access to different neuronal subtypes that may repair damaged nerve circuits with greater efficacy.[8,10] Interestingly, it has been shown that a variety of injuries including TBI stimulate the endogenous reservoir of NPCs in the subventricular zone (SVZ) to proliferate and migrate to the site of injury in adult rodents, primates, and humans.[11–14] Upon arrival in the TBI cortex, these cells primarily differentiate into astrocytes but also a small number of neurons, resulting in minor improvements to motor and sensory function in rodents.[15,16] While naturally-occurring neurogenesis is not enough to restore function following TBI, it motivates research that will enhance the proliferation, migration, and differentiation of SVZ NPCs into cortical neurons.[8,10] Although direct injection of transcription factor-expressing viruses into the injured CNS has shown promise as a means for reprogramming endogenous cells into neurons,[17–20] viral strategies are translationally limited by their immunogenicity, genetic cargo capacity, and complex manufacturing processes in comparison to non-viral methods.[21,22] Thus, we seek to augment the NPC repair response through non-viral transfection of therapeutic genes in SVZ NPCs through intracerebroventricular (ICV) injection of polyplexes.

We have previously demonstrated delivery of reporter plasmids to cells of the SVZ in healthy mice;[23,24] however, very little is known about the impact of TBI on in vivo transfection. In this work, we utilize our most promising polymeric gene carrier (VIPER[24,25], Scheme 1) to deliver various plasmid cargoes through ICV injection in a controlled cortical impact (CCI) mouse model of TBI. Although the clinical etiology of TBI is extremely diverse, CCI offers promise as a translational model that generates reproducible and quantifiable cognitive and motor deficits in mice that mimic human symptoms.[26,27] As a first step towards therapeutic transfection after TBI, we first optimize the timing of transfection post-injury using luciferase reporter plasmids in order to capitalize on the dynamic cellular proliferation response to injury. We next analyze the distribution of GFP transfection after injury in various brain regions contacting the ventricular space through confocal microscopy, and then further investigate extracellular barriers to gene delivery in these regions through a combined in vivo and in vitro approach. This work highlights the significant hurdles between current non-viral transfection approaches and therapeutic transfection after CCI in mice and establishes guidelines for future vector development.

Scheme 1.

Chemical structure of the Virus Inspired Polymer for Endosomal Release (VIPER).

The diblock copolymer is synthesized via RAFT polymerization and subsequently conjugated to a cysteine-modified melittin via disulfide exchange with the PDSEMA monomer. The first block (green) enables sterically shielded nucleic acid condensation and the second block (purple) enables pH-sensitive micellization to encapsulate the lytic peptide until endosomal acidification.

2. Experimental Section

2.1. Material sourcing, polymer synthesis, and polyplex information.

Endotoxin-free plasmid pCMV-Luc™ (ProMega) and pMAX-GFP™ (Lonza) were purified with the Qiagen Plasmid Giga kit (Qiagen) according to the manufacturer’s protocol. EdU (5-ethynyl-2’-deoxyuridine) purchased from Lumiprobe was dissolved at 10 mg/mL in saline by heating at 80 °C for 10 min before storage at −20 °C in 0.22 μm sterile-filtered aliquots until use. All chemicals used for polymer and peptide synthesis were purchased from either Sigma Aldrich or Thermo Fisher Scientific and used without further purification as previously described.[24] The block copolymer p(OEGMA_8.6-co-DMAEMA_50.0)-bl-p(DIPAMA_25.3-co-[PDSEMA-g-melittin]_1.0) termed “VIPER” was prepared and characterized in previous work.[24] It is important to note that VIPER micelles (~25 nm diameter) condense nucleic acids via charge-charge interactions between DMAEMA tertiary amines (N) and nucleic acid phosphates (P) to form polyplexes composed of multiple micelles. When formulated by simple mixing in water at N/P 10, VIPER pDNA polyplexes are ~110–130 nm in diameter in pH 7.4 PBS and display a zeta potential of +15–20 mV in 10 mM NaCl, similar to other polyplexes reported in the literature.[28–32] VIPER polyplexes undergo a pH-dependent disassembly following endocytosis that reveals the conjugated lytic peptide melittin, resulting in endosomolysis and efficient cytoplasmic plasmid delivery.[25]

2.2. Controlled cortical impact (CCI).

All animal procedures were completed according to protocols approved by the Institutional Animal Care and Use Committee at the University of Washington. Moderate CCI was performed on female C57Bl/6 mice (8 wk old) using a custom electromechanical impactor described by the Ohio State University ESCID Contusion Model.[33] Briefly, in this model, a craniotomy is performed by drilling a burr hole (centered 1 mm medial/lateral, −1.5 mm rostral/caudal to Bregma). A 2mm probe attached to the contusion device is lowered onto the dura covering the cortex until contact is visualized by the dampening of force oscillations on an oscilloscope. Brain deformation of 0.8 mm at a velocity of 5 m/sec is performed with a slope of 0.3 V/ms; actual displacement and mean force are recorded for each impact. Bleeding is dried with gauze, gel foam and bone wax is used to patch the burr hole, and staples are utilized for skin closure prior to recovery and analgesia. While the primary necrotic injury occupies the somatosensory and parietal association cortexes, the secondary injury zone comprised of inflammation and disrupted blood-brain-barrier function extends into the hippocampus.[34,35] (Figure S1) This overall moderate injury leads to significant motor and learning deficits that can be reliably evaluated through behavioral assessment.[36]

2.3. In vivo plasmid transfection in the brain.

Intracere (ICV) injection was performed as described before.[29] Polyplex formulations were prepared in 5% glucose solution containing 2.5 μg of plasmid DNA (N/P 10) by vigorously pipetting polymer into DNA and resting the mixture for at least 10 minutes. Female C57/Bl6 mice (8 wk old) were anesthetized by intraperitoneal injection of Avertin (500 mg/kg body weight). After craniotomy, a burr hole (1 mm diameter) was made on the right-side of the skull using a dental drill, and 10 μL of polyplex was stereotaxically injected (Bregma, −0.5mm; Medial/Lateral, 1.0mm; Dorsal/Ventral, 1.8mm) using a 33 gauge Hamilton syringe. The injection was made at 2 μL/min and the syringe was kept in the injection site for 2 min to prevent backflow prior to needle removal. When used, EdU was injected intraperitoneally at a dose of 50 mg/kg 8 h after transfection.

For luciferase plasmid transfections, brain compartments were harvested 48 h after transfection as distinct tissues: Left or Right hemisphere (without olfactory bulbs, and Hindbrain (including cerebellum and brainstem). Each tissue was collected and snap-froze in lysis buffer supplemented with protease inhibitors (Roche). Three freeze-thaw cycles were performed in liquid nitrogen, tissues were mechanically homogenized, and lysate was cleared by centrifugation at 21,000 g for 15 min at 4 °C. Clarified lysate was assayed for luminescence with luciferase substrate (ProMega) using a plate reader and relative light units (RLU) were normalized by protein content as determined by BCA Protein Assay Kit (Pierce). Thus, gene expression was reported as the mean + standard deviation RLU/mg protein for each brain region. Whole brain gene expression was calculated as the sum of RLU/mg values of all regions per brain and then whole brain sums were averaged in each group before analysis.

For GFP plasmid or QD585-labeled luciferase plasmid transfection, mice were euthanized 48 h or 1 h after transfection, respectively, and perfused intracardially with 0.9% saline followed by 4% paraformaldehyde in 0.1 M phosphate buffer. Brains were then processed for histology as described below.

2.4. Histology, confocal microscopy, and image quantification.

After perfusion and fixation, brains were excised and equilibrated to 30% sucrose in phosphate buffer. Brains were embedded in OCT and sectioned into 30 μm-thick coronal slices stored floating in PBS. For immunofluorescent labeling, slices were rinsed with PBS and blocked in PBS, 0.3% Triton X-100, 2% bovine serum albumin (BSA) for 1 h. If performing click chemistry staining of EdU, slices were washed three times with PBS, incubated for 30 m in labeling cocktail (1 μM N₃-Cy5 [Click Chemistry Tools], 1 mM CuSO₄, 100 mM sodium ascorbate), and then washed three times in TBS before proceeding.[37] Primary antibodies (chicken anti-GFAP [1:500, Millipore AB5541]; rabbit anti-Iba1 [1:500, Wako 019–19741]; rabbit anti-NG2 [1:500, Millipore AB5320]) were applied to the tissue sections in PBS, 0.3% TritonX-100, 2% BSA overnight at 4 °C. Sections were rinsed three times for 20min in TBS, 0.1% Tween 20 and species appropriate secondary antibodies conjugated with fluorophore were incubated in PBS, 0.1% Tween 20, and 2% donkey serum for 2 h. Sections were rinsed three times for 20min in TBS–Tween, with the last rinse containing the nuclear marker, 4′,6-diamidino-2-phenylindole (DAPI; 0.5 μg/ml). Sections were then mounted onto glass slides, sealed and cover-slipped with polyvinyl alcohol, and imaged using a custom Leica SP8X confocal scanning laser microscope housed at the W. M. Keck Microscopy Center at the University of Washington Medical Center.

Quantification of transfection in brain slices rostral and caudal to the injection site was performed by a blinded researcher. The location (approximate within ~0.33 mm and assigned to one of the regions shown below) and number of GFP+ cells was recorded from ≥ 15 slices per brain in ≥ 3 brains per group. All cells in the rostral and caudal regions were separately summed for each brain. Regional sums were then averaged across brains and plotted as the mean + SEM for each group.

2.5. Conjugation of luciferase plasmid DNA with QD585.

Luciferase plasmid was labeled with Qdot™ 585 Streptavidin Conjugate (QD585, ThermoFisher) utilizing the DNA-intercalating crosslinker Psoralen-PEO-biotin (ThermoFisher) as previously described with minor protocol modifications.[38] In brief, the psoralen crosslinker (20 mM in DMSO) was diluted 1:100 v/v with luciferase plasmid DNA (2 mg/mL in water) and irradiated using a 50 W longwave UV lamp for 1 h at room temperature. The plasmid was ethanol/acetate precipitated and biotin content verified to be in excess of 1000 biotins/plasmid using the Quant*Tag™ Biotin Kit (Vector Laboratories) and a Nanodrop spectrophotometer (ThermoFisher). Strep-QD585 solution was mixed with biotinylated plasmid at 1:1 molar ratio and incubated at room temperature overnight. Plasmids were ethanol/acetate precipitated to remove unconjugated Strep-QD585 and resuspended in 5% glucose for use in transfection. Conjugation purity was verified by gel electrophoresis and the degree of conjugation was determined to be between 0.5–1 QD585/pLuciferase using the HS dsDNA Qubit assay (ThermoFisher) to quantify DNA concentration and a fluorescence plate reader to quantify QD585 concentration via a custom standard curve.

2.6. Cell culture and in vitro plasmid uptake and transfection.

CHO (K1 wt ATCC# CCL-61; pgsE-606 ATCC# CRL-2246; pgsA-745 ATCC# CRL-2242) were maintained at 37 °C/5% CO₂ in F-12K base media (Gibco) supplemented with 10% fetal bovine serum and penicillin/streptomycin and passaged every three days or when 75% confluent. Cells were seeded at a density of 25,000 cells/well in 24-well plates and incubated overnight before assay. Polyplexes were prepared at N/P 5 with 1 μg QD585-pLuciferase (for uptake studies) or 1 μg pMAX-GFP™ pDNA (for transfection studies) and allowed to rest for at least 10 min. Each polyplex solution was then added dropwise directly to each well (in triplicate). In uptake studies, cells were washed twice with PBS and lifted with trypsin for analysis by flow cytometry at either 2, 4, or 8 h after polyplex addition. In transfection studies, cells were given fresh complete medium after 4 h incubation with polyplexes and analyzed by flow cytometry 48 h after transfection. Singlet cell events recorded on an Attune NxT flow cytometer (ThermoFisher) were gated using untreated cells to determine uptake (QD585+) and transfection (GFP+). All experiments were performed as biological triplicates and variance reported as the standard deviation of the mean.

2.7. Polyplex unpackaging assays.

VIPER or bPEI polyplexes were formulated with pLuciferase at N/P 10 and incubated in the presence of various concentrations of heparin sulfate or chondroitin sulfate sodium salt (Sigma) for 30 min at 37 °C. Samples were then loaded onto a 0.5% agarose gel containing TAE buffer (40 μmM tris-acetate, 1 mM EDTA) and 0.5 μg/mL ethidium bromide, and were electrophoresed at 110 V for 30 min before imaging on a UV transilluminator.

2.8. Statistical analysis.

All statistical analyses were performed in Prism software (Graph Pad Software, La Jolla, CA) using a two-tailed Student’s t-test with unequal variance and Welch’s correction.

3. Results and Discussion

3.1. Luciferase transfection in the brain following controlled cortical impact (CCI).

We first investigated whether overall intraventricular polyplex transfection efficiency was altered by CCI. We hypothesized that the length of time between injury and transfection could dramatically alter transfection efficiency due to both physical changes in brain anatomy that evolve with the primary and secondary injury over time (e.g. edema, inflammation)[3,39] and due to increased mitosis among the neural progenitor cells (NPCs) of the subventricular zone (SVZ).[11] It has been reported that SVZ NPC proliferation is enhanced as soon as 1 day post injury (DPI) and peaks sometime between 3 and 7 DPI in various rodent models of TBI.[11,13,40,41] Because nuclear uptake and expression of plasmid DNA often depends on mitosis,[42] we hypothesized that timing transfection to coincide with peak SVZ NPC proliferation would increase transfection efficiency and eventually therapeutic benefit. Thus, we performed CCI followed by ICV transfection of luciferase plasmid ipsilateral to the injury at various DPI, followed by harvest and in vitro luciferase detection in brain lysates 48 h after transfection.

We observed that CCI tended to increase subsequent transfection at all time points tested relative to transfection in uninjured mice. (Figure 1A) Although increases in transfection efficiency were not statistically significantly different between groups, we did observe a trend of increasing transfection with increased time post injury that peaked at 3 DPI, in agreement with past studies identifying this time point at the peak of SVZ proliferation.[41] The relative distribution of transfection among brain compartments was not altered by TBI, with the lowest transfection observed in the left brain-hemisphere (contralateral to injection) and the highest transfection observed in the hindbrain in all groups. (Figure 1B) This is in agreement with our past data from uninjured mice,[24] and is likely a result of polyplex drainage into the fourth ventricle via the flow of cerebral spinal fluid (CSF).[43–45] Thus, CCI does not dramatically alter ICV transfection, and may even potentiate transfection if performed 3 DPI.

Figure 1. Transfection efficiency of polyplexes injected ICV at various time points after CCI.

(A) Relative luciferase activity in homogenized brains displayed as a sum of all brain regions. (B) Luciferase activity within tissue lysates of separate brain region (Left or Right hemisphere and Hindbrain) were normalized by protein concentration to analyze VIPER-mediated transfection in control, 1, 3, and 7 DPI versus naked DNA only. Data are plotted as mean ± SD where N = 3–4 mice. (*, p < 0.05; **, p < 0.01; ****, p < 0.0001 by ANOVA with Dunnett’s multiple comparisons).

3.2. Distribution of GFP transfection by anatomical location and cell type.

Encouraged by these results, we next sought to determine whether CCI influences the cell types that are transfected in specific anatomical locations in the brain. Thus, we performed intraventricular delivery of polyplexes in both injured and uninjured mice using a plasmid encoding GFP, chased transfection with an intraperitoneal injection of EdU to label mitotic cells (in S-phase) after 24 hours post-injury. Since we observed poor ICV transfection (i.e. luciferase activity) at 7 DPI, we used confocal microscopy to analyzed GFP-transfection at 1 or 3 DPI versus uninjured mice (Figure 2). As shown, VIPER transfected cells within proliferative zones (i.e. SVZ) enriched with EdU+ cells after injury. Interestingly, the proportion of GFP+ EdU-labeled cells was low. These observations suggests that VIPER polyplexes may have a short resident tissue time and requires the presence of actively dividing cells versus cells at the start of S-phase (Figure 2A–C). While TBI did not drastically reduce transfection (as shown above, Figure 1), the overall number of GFP-transfected cells was less than in previous studies by our group with other polymers.[23]

Figure 2. Immunohistochemistry and EdU click-labeling reveal that CCI does not dramatically change proliferation or transfection in the SVZ.

Confocal micrographs of the SVZ region following transfection with plasmid encoding GFP and fluorescent staining for GFAP and EdU. Mice were transfected without injury (A), 1 DPI (B), or 3 DPI (C), EdU was administered IP 8 h after transfection, and mice were sacrificed and perfused 48 h after transfection. Images are representative of results from N = 4 mice per group. Scale bar = 50 μm.

In qualitative agreement with luciferase transfection data, we observed slightly more GFP+ cells in mice transfected 3 DPI than in mice transfected 1 DPI or without injury, with the greatest frequency of transfection observed in the fimbria and at the base of the hippocampus within 1 mm of the injury on the rostral-caudal axis independent of injury. (Figure 3, Figure S2) The majority of transfected cells were observed at interfaces of tissue and CSF (e.g. fimbria and ventricle [Figure 3A]; at the hippocampal/thalamic fissure [Figure 3B]), indicating that polyplexes did not penetrate tissue, which limited transfection to the ventricular surfaces caudal to the injection. Quantification confirmed higher numbers of GFP+ cells in slices caudal to the injection site (−0.66 to −2 mm Bregma) compared to rostral slices (+0.33 to −0.66 mm Bregma), with the highest level of GFP transfected cells 3 DPI. (Figure 3) While not statistically significant, these patterns recapitulate the trends identified by luciferase transfection and highlight the effects of CSF circulation observed by other studies.[46–49]

Figure 3. Transfection observed primarily in specific anatomic regions at the CSF-tissue interface.

Confocal micrographs of the fimbria (A; near injection site) and the base of the hippocampus (B; caudal to injection) following transfection 3 DPI with plasmid encoding GFP and fluorescent staining for GFAP and Iba1. Scale bar = 50 μm. Quantification of transfection in brain slices rostral and caudal to injection site showed more transfected cells in caudal regions; however, no significant differences between brain regions or injury timing were observed.

We observed heterogeneous transfection of various cell types dependent on the region investigated but independent of injury. For example, we observed transfection of morphologically complex cells resembling oligodendrocytes in the fimbria in all mice (Figure 3A) but were unable to identify the cells that were consistently transfected at the base of the intrapyramidal granule cell layer of the dentate gyrus (DG) in the septal hippocampus (Figure 3B). Although we did observe transfection of GFAP+ astrocytes, in no cases were transfected cells Iba1+ microglia. (Figure S3) In summary, although the incidence of GFP-expression was much lower than we expected, we observed that only select brain structures are transfected following ICV injection and that CCI does increase the number of cells transfected at these sites.

3.3. Quantum dot labeling reveals extracellular barriers to plasmid uptake.

Drawing on our experience investigating extracellular barriers to gene delivery following intravenous injection, we hypothesized that poor cellular uptake or premature polyplex unpackaging could also limit transfection in the ventricular space. We have previously shown that some cationic polymers will forgo the condensation of plasmid DNA in favor of electrostatic interactions with negatively charged extracellular matrix (ECM) components (e.g. glycosaminoglycans) and limit intracellular plasmid uptake.[38] In that work, we labeled plasmids with a small number of ultra-bright quantum dots (QDs), which enables the detection of individual plasmids delivered by polyplexes in vivo without drastically altering polyplex properties.[50] We transfected QD-plasmid conjugates in healthy mice to better characterize the localization of plasmid cargo following intraventricular polyplex injection. We observed similar regional patterns in plasmid distribution as observed in GFP transfection, with most QD-plasmid found in the fimbria. (Figure 4A) High magnification imaging of plasmid found in the fimbria revealed both diffuse and fibrous fluorescent signal which indicates a mixture of intracellular and extracellular plasmid. (Figure 4B) Interestingly, all tissue within a ~200 μm radius of QD-plasmid displayed very little immunofluorescent staining for NG2 proteoglycan and created a “halo” of limited NG2-staining in the tissue proximal to GFP-cells. We previously observed that fluorescently-labeled polyethylenimine binds to liver ECM after systemic administration.[38] Thus, we rationalized that “halo” around GFP-expressing cells could be the result of electrostatic coating of sulfated glycosaminoglycan domains with VIPER chains that prevents antibody staining. Taken together, these observations suggest that VIPER’s transfection is limited by ECM components in the brain that promote polyplex unpackaging.

Figure 4. Plasmid delivery patterns mimic regional patterns of transfection and are driven by electrostatic interactions with sulfated proteoglycans.

Confocal micrographs of serial tissue sections at low (A) and high (B) magnification of the fimbria following transfection with QD585-conjugated luciferase plasmid and fluorescent staining for NG2 chondroitin sulfate proteoglycan. Scale bar = 200 μm.

3.4. Plasmid transfection and uptake in ECM mutant cell.

In order to examine how proteoglycans in the ECM affect VIPER, we next performed in vitro GFP plasmid transfection with Chinese hamster ovary (CHO) cells with mutations in various glycosaminoglycan (GAG) synthetic pathways: CHO-pgsA-745 and CHO-pgsE-606 cells. As demonstrated in previous studies, CHO-pgsA-745 and pgsE-606 cells have altered xylosyl-transferase activity and heparin sulfate N-sulfotransferase, respectively.[51,52] While, each mutant displays changes in binding and uptake of molecules, each cell-type demonstrates consistent and functional endocytosis.[53–55] Thus, we compared GFP-expression and Qdot uptake in CHO-K1, proteoglycan deficient CHO-pgsA-745 cells, and CHO-pgsE-606 cells that lack proteoglycan sulfation.[51,52] Using flow cytometry, we demonstrate that VIPER transfects wt and pgsE-606 cells with similar efficiency at N/P 5 (36% vs 46% GFP+), but transfection is significantly decreased in pgsA-745 cells (3% GFP+). (Figure 5A)

Figure 5. In vitro plasmid transfection and uptake in GAG mutant CHO cell lines.

Flow cytometry was used to quantify GFP+ cells 48 h after pMAX-GFP polyplex addition (A) or to quantify the percentage of QD585+ cells at various times after QD585-pLuciferase polyplex addition (B). The average median fluorescent intensity (MFI) of QD585 signal in single cells increased with uptake over time (C). Data are plotted as the mean ± SD of > 3 experiments and statistical significance derived from Student’s t-test (* p < 0.05; ** p < 0.01).

To determine whether transfection efficiency was dictated by plasmid uptake, we next performed mock-transfections using polyplexes formulated with QD585-pLuciferase and quantified intracellular QD585+ signal by flow cytometry. (Figure 5B) Accordingly, plasmid uptake increased with polyplex incubation time for all cell types. However, wt and pgsE-606 cells demonstrated near total uptake at 4 h (72% and 76%, respectively), while only a small fraction of pgsA-745 cells demonstrate plasmid uptake after 8 h incubation (17%). Moreover, the overall shift in QD585 median fluorescence intensity (MFI) from 0–8 h was significantly lower in pgsA-745 cells (6.3-fold) compared to wt and pgsE-606 cells (16.5- and 20.5-fold, respectively), indicating that the total number of plasmids taken up by pgsA-745 cells was low. (Figure 5C; Figure S4) These data demonstrate that VIPER polyplex uptake is enhanced by electrostatic proteoglycan interactions at the cell surface and that polyplex uptake efficiency dictates transgene expression. Furthermore, we suggest that VIPER polyplexes may also be attracted to unproductive proteoglycan interactions with GAG-rich ECM that could hamper CNS tissue penetrance in vivo.

The charge density, pKa, and molecular weight of both GAGs and polycations are known to have differentially positive and negative effects on polyplex transfection.[56–58] As demonstrated by previous studies, GAGs are required for polyplex uptake,[56] yet heparin sulfate proteoglycan can lead to premature polyplex unpackaging by out-competing DNA binding with polycations.[57,58] In this study, we observed a drastic reduction of VIPER uptake and transfection in GAG deficient cells, but transfection and uptake are not adversely affected in pgsE-606 cells lacking proteoglycan sulfation. In order to determine if sulfate interactions could lead to polyplex unpackaging, we incubated VIPER or bPEI polyplexes (N/P 10) with varying concentrations of either heparan sulfate or chondroitin sulfate. (Figure 6) While neither VIPER nor bPEI polyplexes were unpackaged by chondroitin sulfate (5 mg/mL), both VIPER and bPEI polyplexes were unpackaged at 100 μg/mL heparan sulfate, which corroborates prior experiments that demonstrated charge density may differentiate GAG-polyplex interactions.[57] Taken together with in vivo NG2 staining (Figure 4) and in vitro transfection data (Figure 5), these results indicate that VIPER requires GAGs for transfection, but that the highly GAG-rich ECM of neural tissue may limit VIPER penetration or cause unpackaging in vivo. Future work will be needed to determine which GAG interactions dominate VIPER transfection in vivo, and whether polycations with different amine functional groups (i.e. primary or secondary amines)[31] or charge densities[32] would better suited for CNS transfection.

Figure 6. Polyplex unpackaging by various sulfated ECM components.

VIPER or bPEI polyplexes were formulated with pLuciferase at N/P 10 and incubated in the presence of various concentrations of heparan sulfate or chondroitin sulfate sodium salt for 30 min at 37 °C. Samples were then loaded onto a 0.5% agarose gel containing TAE buffer and ethidium bromide, and were electrophoresed at 110 V for 30 min before imaging on a UV transilluminator.

4. Conclusion

Herein we present the first investigation of intraventricular polyplex transfection following controlled cortical impact in mice. Using a custom polymer we previously reported to yield high luciferase plasmid expression in the brain, we show that CCI slightly increases overall transfection efficiency and that the distribution of transfection throughout the major compartments of the brain is unaffected by CCI, but polyplexes do not penetrate deep into brain tissue. We demonstrate that this pattern of distribution is not dependent on injury but may be a direct result of interactions between VIPER and ECM proteoglycans. Given that chondroitin sulfate proteoglycan upregulation has been implicated as a potential therapeutic target in TBI, this work motivates future polymer designs that can take advantage of charge-charge interactions for polyplex targeting without sacrificing plasmid uptake, perhaps by increasing polycation charge density or altering polycation pKa. Because TBI is known to affect cellular genesis, CSF flow, and CSF ionic composition, the impacts of these variables on non-viral transfection represent additional valuable areas of future study to improve vector design. In summary, we report that polyplex-mediated plasmid transfection is largely unaffected by CCI in mice but that extracellular barriers to delivery must be accounted for in order to transfect therapeutically relevant numbers of cells in the intraventricular space.

HIGHLIGHTS:

• Polymer-mediated gene transfer efficiency to the brain is not inhibited by injury.

• Maximal VIPER mediated gene delivery occurs in the brain three days post-injury.

• CNS glycosaminoglycans are a likely barrier to polyplex-mediated gene delivery.