PBAE/DNA Nanoparticles Are Effective in Btics from Multiple Patient Sources

The purified polymers were evaluated on a second sample of primary cultured BTICs derived from a different patient (JHGBM-276), using GFP DNA as a transfection marker (Figure S1, Supporting Information). Similarly high transfection efficacy was seen in JHGBM-276 BTICs, with PBAE 447e once again a leading polymer (61 ± 3% transfection with 97 ± 7% viability). However, PBAE 537e was found to be the top formulation for transfection of JHGBM-276 BTICs, with up to 76 ± 2% transfection with 90 ± 4% viability, although it transfected only between 20 and 45% of cells in JHGBM-551 BTICs. A moderate correlation in transfection efficacy of PBAE formulations (r² = 0.42) was found between two oncosphere cultures (JHGBM-551 and JHGBM-276), with 537e as the only statistical outlier. Outlier statistics calculations were done using Prism software (see the Methods). The non-537e polymers show a stronger correlation between the two primary glioblastoma oncospheres cultures (r² = 0.66) (Figure ​Figure33). A strong correlation was also found between transfection of oncospheres and monolayer cultures (r² = 0.79), indicating that, while a few polymers like 537e may have some specificity for one human cell sample over another, trends in transfection found in one GBM cell sample are largely applicable to other GBM samples, even when derived from separate patient sources.

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Figure 3

PBAE nanoparticles transfect BTIC oncospheres and monolayers from different patient sources. (A) Fluorescence and brightfield micrographs showing transfection of JHGBM-551 and JHGBM-276 primary cultures with PBAE 447e, effective for transfection of both cell types, and PBAE 537e, more effective for transfection of JHGBM-276 than JHGBM-551. In each pair of images, (top) brightfield and transgene signal merged (DsRed or GFP, false-colored green for both) and (bottom) DsRed or GFP only. (B) Correlation between transfection efficacy of all PBAEs and JHGBM-551 and JHGBM-276 oncospheres (black line) and for comparison without including outlier PBAE 537e (gray line). Points on the graph are labeled with the PBAE used to transfect them. (C, D) Transfection in 2-D (monolayer) and in 3-D (oncospheres) shows high correlation (r² = 0.79, 0.64). Graphs show mean ± standard error of the mean (SEM).

Nanoparticle Transfection Efficacy Is Specific for Human Brain Tumor Initiating Cells over Human Neural Progenitor Cells

Our previous work suggested that an optimized PBAE nanoparticle formulation could be more effective at transfecting one patient-derived BTIC culture, JHGBM-551, than one fNPC culture, F34.²⁹ These observations were significantly strengthened by evaluating additional PBAE nanoparticle formulations in multiple patient-derived BTIC cultures and multiple fNPC cultures. Here, our top polymeric nanoparticle was tested on four patient-derived BTIC cultures (JHGBM-276, JHGBM-551, JHGBM-854, and JHGBM-965) as well as on three healthy, noncancerous primary human fetal neural progenitor cell cultures (fNPCs) (F34, F54, and F48) cultured in the same conditions as their cancer counterparts. We first evaluated PBAEs in JHGBM-276 BTICs and F34 fNPCs (Figure ​Figure44A), which showed that many of the PBAEs had significantly higher transfection in BTICs compared to fNPCs. We further evaluated our top PBAE, 447e, on the four BTIC and three fNPC cultures using two PBAE/DNA ratios (30 w/w and 60 w/w) and a range of nanoparticle doses. When results of transfection were combined for all BTIC and fNPC cells, at every dose tested, significantly higher expression was seen in BTICs over fNPCs (Figure ​Figure44B,C). Of note, the difference between BTIC and fNPC transfections was even more striking when measured by intensity of GFP fluorescence from the cell population, indicating that several times more protein was being produced by transfected BTICs compared to transfected fNPCs. This was particularly evident at lower doses, for which fNPCs were transfected very poorly or not at all, while most of the BTIC samples still showed significant transfection. Fluorescence microscopy qualitatively confirmed these results (Figure ​Figure44D). The specificity of transfection at low doses has great implications for an in vivo application.

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Figure 4

PBAE nanoparticles as GBM-specific gene delivery vehicles. JHGBM-276 BTICs and F34 fNPCs were transfected with leading PBAE formulations. (A) Several polymers showed a higher transfection efficacy in JHGBM-276s cells when compared with healthy cells. (B, C) BTICs were more efficiently transfected with top polymer (447e, tested at 30 and 60 w/w) than fNPCs at equivalent w/w ratios and nanoparticle doses, measured by percent of cells transfected and GFP fluorescence intensity. (D) Fluorescence micrographs qualitatively show the same result. For all graphs, *p < 0.05, **p < 0.01, ***p < 0.001. Graphs show mean ± standard error of the mean (SEM).

To determine if cell division rate was the determinant factor in the specificity of our polymers, we compared the cellular growth rates as well as the cellular uptake of nanoparticles in each primary culture of BTICs and fNPCs. Cellular growth rate can affect gene delivery, as the nuclear membrane is a significant delivery barrier preventing exogenous plasmid DNA from being able to be successfully transcribed. Cells that quickly divide quickly can reduce the impact of this barrier and increase the likelihood of successful transfection. There was no statistically significant difference between the cellular doubling times of BTICs and fNPCs, indicating cell division rate was not a factor in this observation (Figure S2, Supporting Information).

We then evaluated if particle uptake, an important step for particle-mediated intracellular gene delivery, was different between BTICs and fNPCs. There was no statistically significant difference between cellular uptake of nanoparticles in fNPCs compared to BTICs (Figure S2, Supporting Information). This result was obtained both when measuring uptake as the percent of cells that are positive for uptake of particles containing fluorescently labeled DNA as well as by total relative fluorescence from uptake of these particles. Even though transfection efficacy was much higher in the BTICs than the fNPCs, the fNPCs had a slightly higher median measure of cellular uptake (relative fluorescence units) compared to the BTICs. These findings indicate that factors other than overall particle uptake and cell division rate are the major contributors to the increased transgene expression seen in BTICs cells compared to healthy fNPCs.

Lyophilized DNA/PBAE Nanoparticles Retain Full Function for at Least 2 Years in Storage

Long-term storage of nanoparticles is necessary to increase the translational potential of this method. In preparation for in vivo studies, we freeze-dried nanoparticles after formation to allow them to be stored stably as well as more easily concentrated for administration of high doses at low injection volume. Previous work using 447e showed that DNA/PBAE nanoparticles lyophilized with sucrose as a protectant retained full transfection efficacy after being stored for 3 months at 4 °C, although efficacy decreased to ~50% after 6 months of storage (Figure ​Figure55). To further determine the shelf life of PBAE nanoparticles, we evaluated lyophilization with different added amounts of sucrose (0–45 mg/mL) and different storage conditions (room temperature ~25, 4, and −20 °C) over a time period of 2 years. While nanoparticles without added sucrose rapidly lost efficacy, nanoparticles formulated at 30 and 45 mg/mL sucrose maintained full efficacy for at least 2 years when stored at −20 °C (Figure ​Figure55).

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Figure 5

Efficacy of lyophilized nanoparticles after long-term storage. (A) Lyophilized nanoparticles of 447e and GFP DNA retained 100% efficacy after 2 years of storage at −20 °C with 30 mg/mL or more of sucrose. Fluorescence micrographs show GFP signal from JHGBM-319 cells after transfection. (B) 447e/DNA nanoparticles retained 100% efficacy after lyophilization with 30 mg/mL or more of sucrose and storage at −20 °C for at least 2 years, quantified by flow cytometry. Graphs show mean ± standard error of the mean (SEM).

Physical and Chemical Characteristics of Nanoparticles Are Conducive to Intracellular Delivery

The polymer 447e, 1.1:1 had a number-average molecular weight of 11.3 kDa and weight-average molecular weight of 36.8 kDa, measured by gel permeation chromatography (GPC). Nanoparticles were made by complexing 447e and DsRed DNA at 60 w/w, lyophilized with 30 mg/mL sucrose, and stored at −20 °C until use. After resuspending the particles in water, we characterized them by nanoparticle tracking analysis (NTA), which measured a mean number-weighted hydrodynamic diameter of 143 nm in PBS (44 nm standard deviation), and by dynamic light scattering, which measured a zeta potential of +16.4 mV (0.96 mV standard error of the mean of five measurements). We have recently shown successful in vivo ophthalmic gene delivery using lyophilized nanoparticles reconstituted at high concentration.³⁰ Here, we evaluated whether our frozen nanoparticles maintained in vivo transfection efficacy after reconstitution and evaluated efficacy in an orthotopic tumor model for the first time. We used 447e, 1.1;1, lyophilized, stored for 1–12 weeks at −20 °C, and then reconstituted just prior to in vivo injection.

Materials

Lipofectamine 2000 (Invitrogen, Carlsbad, CA), Opti-MEM I (Invitrogen), pEGFP-N1 DNA (Elim Biopharmaceuticals, Hayward, CA), pDsRed-Max-N1³⁹ (Addgene DNA plasmid 21718, Cambridge, MA), and cell culture media components were used as received. Monomers used for synthesizing polymers (Scheme 1) were purchased as follows: 1,3-butanediol diacrylate (B3b; Sigma-Aldrich, St. Louis, MO); 1,4-butanediol diacrylate (B4; Alfa Aesar, Ward Hill, MA); 1,5-pentanediol diacrylate (B5; Monomer-Polymer and Dajac Laboratories, Trevose, PA); 3-amino-1-propanol (S3; Alfa Aesar); 4-amino-1-butanol (S4; Alfa Aesar); 5-amino-1-pentanol (S5; Alfa Aesar); 1,3-diaminopropane (E1; Sigma-Aldrich); 1,3-diaminopentane (E3; TCI America, Portland, OR); 2-methyl-1,5-diaminopentane (E4; TCI America); 1,11-diamino-3,6,9-trioxaundecane (E5; TCI America); 2-(3-aminopropylamino)ethanol (E6; Sigma-Aldrich); 1-(3-aminopropyl)-4-methylpiperazine (E7; Alfa Aesar); 1-(3-aminopropyl)pyrrolidine (E8; TCI America); and cystamine dihydrochloride (E10; Alfa Aesar). 4′,6-Diamidino-2-phenylindole dihydrochloride (DAPI) was purchased from Sigma and used as a 750 nM solution in PBS. Other materials were reagent grade.

Polymer Synthesis

PBAEs were synthesized according to previously reported protocols.²¹ For initial screening, one diacrylate backbone (“B”) monomer was mixed with one monoamino side chain (“S”) monomer at a 1.2:1 molar ratio, with B in excess to ensure that the B–S base polymer would be diacrylate-terminated. The mixture was stirred at 90 °C for 24 h. The resulting base polymer was then dissolved in anhydrous dimethyl sulfoxide (DMSO). A 0.5-M solution of one end-cap (“E”) small molecule in DMSO was added for a final base polymer concentration of 100 mg/mL, and the mixture was vortexed for 1 h at room temperature. Polymers were stored until use at 4 °C in small aliquots to reduce the number of freeze–thaw cycles.

Top polymers were chosen from the initial transfection screening done in JHGBM-551 BTICs. These polymers were resynthesized in a purified form as described previously at B:S ratios ranging from 1.05:1 to 1.2:1.³⁶ Briefly, the base polymer and end-cap were dissolved in anhydrous tetrahydrofuran (THF) instead of DMSO. After 1 h of stirring at room temperature, the polymers were precipitated into anhydrous diethyl ether. The precipitate was collected by centrifugation, washed again with ether, and centrifuged to isolate the end-capped polymer. Residual ether was removed under vacuum for 48 h. The dry PBAEs were then dissolved in anhydrous DMSO (100 mg/mL) and stored at −20 °C in small aliquots until use.

Molecular weight of the top polymer was measured by gel permeation chromatography (GPC; Waters, Milford, MA) in BHT-stabilized tetrahydrofuran with 5% DMSO and 1% piperidine. Number-averaged and weight-averaged molecular weight (M_n and M_w, respectively) were calculated using polystyrene standards.

Cell Culture

All the primary cultures of BTICs studied here were obtained from adult patients undergoing surgery for GBM (Table 1), after informed consent, following institutionally approved protocols. Samples were prepared and maintained as previously described.⁴⁰ Cells were maintained in culture as nonadherent oncospheres in complete BTIC medium [DMEM/F-12 (1:1) with 1X B-27 supplement, 1% antibiotic–antimycotic (Invitrogen), 20 ng/mL basic fibroblast growth factor (bFGF), and 20 ng/mL epidermal growth factor (EGF)]. For passaging, oncospheres were collected by centrifugation and were mechanically dissociated by tituration. Primary cultures of human fetal neural progenitor cells (fNPCs) F34, F48, and F54 were obtained as described previously.^29,41 Briefly, following approval by the Johns Hopkins University Institutional Review Board, intraoperative human central nervous system (CNS) tissues were obtained at 17 weeks of gestation, which were obtained following written informed consent for clinical procedures, were used for this research since they were considered to be pathological waste. Brain cortical tissue was mechanically dissociated and cells were maintained in 2:1 high-glucose DMEM (Invitrogen)/Ham’s F-12 (Cellgro), 1X B-27, 1% anti–anti, 20 ng/mL bFGF, 20 ng/mL EGF, 20 ng/mL leukemia inhibitory factor (LIF, Millipore, Billerica, MA), and 5 μg/mL heparin (Sigma)].

Preparation of Cy3-Labeled DNA

eGFP DNA was labeled with Cy3 using a Label IT Tracker kit (Mirus Bio) according to the manufacturer’s instructions. Briefly, a solution of 125 μg/mL DNA and 100 μL/mL Label IT Tracker reagent was prepared in buffer and then incubated at 37 °C for 3 h on an orbital shaker. The labeled DNA was precipitated with ethanol in 300 mM sodium acetate buffer and cooled at −20 °C for 30 min. The DNA was pelleted by centrifugation at 15000g at 4 °C for 15 min, washed with 70% ethanol, and centrifuged again. The dried pellet was reconstituted in fresh unlabeled eGFP DNA in water. The concentration was verified using by absorbance at 260 nm using a Nanodrop 2000 with associated software (v. 1.4.1) (Thermo Fisher, Waltham, MA). The absence of protein from the solution was measured by absorbance at 280 nm (A₂₆₀/A₂₈₀ ≥ 1.7). The labeled DNA was diluted further with unlabeled nucleic acid to a final dye-to-nucleotide molar ratio of 1:350.

Transfection Experiments in Vitro

For 3-D transfections, BTIC oncospheres 200–1000 μm in diameter were collected by centrifugation and resuspended in complete BTIC medium without dissociation using a 5 mL serological pipet. A small aliquot was removed, mechanically dissociated with a 200 μL micropipet, and used for counting and determination of cell density. Cells were seeded into round-bottom, nontissue culture-treated 96-well plates at 1.5 × 10⁴ cells/well in 100 μL complete culture medium and incubated at 37 °C overnight.

For nanoparticle preparation, DsRed (for GFP⁺ JHGBM-551 cells) or eGFP DNA (for all other unlabeled cells) was diluted to 60 μg/mL in 25 mM sodium acetate pH 5 buffer (NaAc). PBAEs were diluted from their stock solutions in DMSO in 25 mM NaAc and added to DNA solutions at PBAE/DNA mass ratios (w/w) of 30, 60, or 90. The resulting mixture was mixed by pipetting and incubated at room temperature for 10 min to allow nanoparticles to form. Nanoparticles (20 μL) in NaAc were added directly to the oncospheres in culture medium in 96-well plates (final nanoparticle/medium ratio of 1:5, final DNA concentration of 5 μg/mL or 600 ng/well, final polymer concentration of 150–450 μg/mL). Oncospheres were incubated with particles at 37 °C for 2 h. A replicate of each plate was transfected in the same way for viability measurements. The plates were then centrifuged at 180g for 5 min. The media and remaining nanoparticles were removed and the oncospheres gently resuspended in complete culture medium. Viability was defined as the metabolic activity retained in each well following transfection, measured after 24 h by MTS (Cell Titer AQueousONE, Promega, Madison, WI) according to the manufacturer’s instructions. Transfection efficacy was measured by fluorescence microscopy and by flow cytometry after 48 h using an Accuri C6 with a Hypercyt high-throughput robotic sampler (Intellicyt) after mechanically dissociating the oncospheres to single-cell suspensions. Transfection data were analyzed with FlowJo 7 software (Treestar). Experiments were done with n = 4 replicates.

After initial screening on JHGBM-551 oncospheres, purified polymers were used for all further experiments. Following a transfection screening using purified polymers, one optimized B/S synthesis ratio was chosen for each PBAE based on high transfection (percent of cells positive for transgene) and low loss in metabolic activity (MTS assay).

For 2-D transfections, a similar protocol was used. Flat-bottom, tissue-culture-treated 96-well plates were coated with 20 μg/mL laminin (Sigma) by incubation at room temperature for 1 h and then washing once with 1xPBS with Ca²⁺ and Mg²⁺. Oncospheres were collected by centrifugation, and the whole pellet was mechanically dissociated with a 200 μL pipet. Dissociated cells were then seeded at 1.5 × 10⁴ cells/well in 100 μL of complete medium into laminin-coated plates. Cells were incubated overnight at 37 °C. For fNPCs, medium was replaced with complete BTIC medium immediately before transfection to ensure that any media interactions with nanoparticles were comparable for all the primary cultures tested. After transfection with DNA/PBAE nanoparticles and incubation for 2 h, the medium and nanoparticles were removed and replaced with fresh BTIC or fNPC culture medium. Viability and transfection measurements were taken in the same way. For flow cytometry, cells were first trypsinized, resuspended in PBS with 2% FBS, and transferred to round-bottom 96-well plates for use with the Hypercyt reader.

Comparison of Fetal and GBM Cell Behavior in Vitro

Uptake of DNA-containing nanoparticles by fNPCs and BTICs was compared. Transfections were then carried out as above on monolayer cultures using fluorescently labeled DNA, keeping cells out of direct light to avoid photobleaching. Uptake was measured by flow cytometry immediately after 2 h of incubation with labeled nanoparticles.

To measure growth rate, BTICs or fNPCs were seeded into laminin-coated 96-well plates at 5000 cells/well and allowed to adhere overnight. At set time points, the metabolic activity of a set of wells was measured by MTS assay (Cell Titer). The doubling time for each BTIC and fNPC culture was calculated by fitting the data to an exponential growth curve.

Formulation and Testing of Lyophilized DNA Nanoparticles

To test the stability of lyophilized particles over time, eGFP DNA was complexed with 447e, 1.2:1, as described previously.²⁹ Briefly, plasmid DNA and 447e were mixed in 25 mM NaAc at a PBAE:DNA ratio of 30 w/w. After particle formation, a solution of d-sucrose in 25 mM NaAc was added for a final DNA concentration of 15 μg/mL and final sucrose concentration of 0, 15, 30, or 45 mg/mL. Nanoparticles were freeze-dried and stored at room temperature (rt), 4 °C, or −20 °C in small aliquots for up to 2 years.

To test the efficacy of stored nanoparticles at various time points, GBM cells (sample JHGBM-319) were seeded in 24-well plates in complete astrocyte medium at 7.5 × 10⁴ cells/well in 500 μL volume. The cells were left overnight to adhere. The next day, nanoparticles were taken from storage and were resuspended in sterile water by pipetting to a final DNA concentration of 15 μg/mL. At the same time, fresh nanoparticles were prepared; briefly, fresh eGFP DNA was diluted in 25 mM NaAc at 30 μg/mL. PBAE 447e, stored in small aliquots at room temperature, 4 °C, or −20 °C, was dissolved in 25 mM NaAc and added to the DNA solution at 30 w/w for final DNA concentration of 15 μg/mL. of reconstituted lyophilized or fresh nanoparticles (100 μL) were then added dropwise to 500 μL of complete astrocyte medium in each well (1.5 μg DNA/well). After 2 h incubation at 37 °C, media and nanoparticles were aspirated from each well and replaced with fresh complete astrocyte medium. Viability and transfection efficacy were measured by MTS assay and flow cytometry as described above and by fluorescence microscopy as described previously.²⁹

Nanoparticle Physicochemical Analysis

The top lyophilized particle formulation was sized using nanoparticle tracking analysis (NTA) using an NS500 (NanoSight, Ltd.). The zeta potential was measured using a Zetasizer Nano NS (Malvern). For both, nanoparticles were formed in 25 mM NaAc at the same concentration as used for transfections, then diluted in 1xPBS as necessary for sizing purposes.

In Vivo Testing of DNA Nanoparticles

Male nude athymic mice were injected with 1 μL of GFP⁺ JHGBM-276 cells in PBS (10⁵ cells/μL) at 8 weeks old. After a 4- or 9-week period for tumor formation, nanoparticles composed of DsRed plasmid DNA complexed with 447e, 1.1:1, at 60 w/w, lyophilized with 30 mg/mL sucrose, and stored at −20 °C were resuspended in water. For particle localization studies, DsRed-coding DNA was first labeled with Cy5 using a Label IT Tracker kit (Mirus Bio) before complexation with PBAE. Nanoparticles (2 μL, 2 μg DNA) were stereotatically injected into the tumor of the mice. Controls included mice injected with nanoparticles but without tumor, mice with tumor but no treatment, and mice with tumor and injected with 2 μg naked DNA in a sucrose solution of the same concentration. After 48 h, mice were anaesthetized with ketamine/xylazine, sacrificed, and fixed by perfusion with PBS and 10% formalin. Brains were removed, stored overnight in 10% formalin, soaked in 30% sucrose in 1 × phosphate buffer for 24 h, and then embedded in Optimal cutting temperature compound (OCT). Embedded brains were snap frozen in a dry ice/ethanol bath and stored at −80 °C until use. Brains were cryosectioned in 10-μm slices and mounted onto glass slides with DAPI and stored at 4 °C, protected from light, until use. Slices were imaged by fluorescence microscopy to assess transfection using an Axio Observer A1 and captured with Axiovision software.

Image Analysis of Brain Sections

The degree of transfection was assessed by image analysis. For mice with tumors that developed over 9 weeks, ImageJ was used to find the area of DsRed fluorescence in each section. ImageJ is a public domain program and was downloaded from http://imagej.nih.gov/ij/. The volume of transgene expression was estimated by multiplying the area of DsRed signal on each slice by the thickness of the slice and adding the resulting volumes. For mice with tumors that developed over only 4 weeks, the Colocalization Colormap plugin on ImageJ was used to find the degree of colocalization of the DsRed and GFP signals based on the method described in Jaskolski et al.³¹ The number of DsRed⁺ pixels that were colocalized with GFP signal was compared with DsRed⁺ pixels not colocalized with GFP. The same analysis was also carried out with Cy5 and GFP signal to compare pixels positive on both channels to pixels positive for Cy5 but not for GFP.

This work was supported in part by the National Institutes of Health (1R01EB016721 and R21CA152473) and MSCRF-TEDCO. We thank Xiaobu Ye for help with statistical analysis. S.Y.T. thanks the National Science Foundation for fellowship support.