A Mixed-Surface Polyamidoamine Dendrimer for in Vitro and in Vivo Delivery of Large Plasmids

pharmaceutics

Article A Mixed-Surface Polyamidoamine Dendrimer for In Vitro and In Vivo Delivery of Large Plasmids

Bhairavi Srinageshwar 1,2,3 , Maria Florendo 1,2, Brittany Clark 4, Kayla Johnson 4, Nikolas Munro 2,3, Sarah Peruzzaro 2,3, Aaron Antcliff 1,2,3, Melissa Andrews 1,2,3, Alexander Figacz 1,2, Douglas Swanson 4, Gary L. Dunbar 2,3,5,6, Ajit Sharma 4,* and Julien Rossignol 1,2,3,* 1 College of Medicine, Central Michigan University, Mount Pleasant, MI 48859, USA; [email protected] (B.S.); fl[email protected] (M.F.); [email protected] (A.A.); [email protected] (M.A.); fi[email protected] (A.F.) 2 Program in Neuroscience, Central Michigan University, Mount Pleasant, MI 48859, USA; [email protected] (N.M.); [email protected] (S.P.); [email protected] (G.L.D.) 3 Field Neurosciences Institute Laboratory for Restorative Neurology, Central Michigan University, Mt. Pleasant, MI 48859, USA 4 Department of Chemistry and Biochemistry, Central Michigan University, Mount Pleasant, MI 48859, USA; [email protected] (B.C.); [email protected] (K.J.); [email protected] (D.S.) 5 Department of Psychology, Central Michigan University, Mount Pleasant, MI 48859, USA 6 Field Neurosciences Institute, St. Mary’s of Michigan, Saginaw, MI 48604, USA * Correspondence: [email protected] (A.S.); [email protected] (J.R.); Tel.: +1-989-774-3303 (A.S.); +1-989-774-3405 (J.R.) Received: 12 June 2020; Accepted: 30 June 2020; Published: 3 July 2020

Abstract: Drug delivery to the brain is highly hindered by the presence of the blood–brain barrier (BBB), which prevents the entry of many potential drugs/biomolecules into the brain. One of the current strategies to achieve gene therapy for neurodegenerative diseases involves direct injection of a viral vector into the brain. There are various disadvantages of viral vectors, including limitations of cargo size and safety concerns. Nanomolecules, such as dendrimers, serve as an excellent alternative to viral delivery. In this study, as proof-of-concept, we used a surface-modified dendrimer complex and delivered large plasmids to cells in vitro and in vivo in healthy rats via intracranial injection. The dendrimers were biodegradable by chemicals found within cells and toxicity assays revealed that the modified dendrimers were much less toxic than unmodified amine-surface dendrimers. As mentioned in our previous publication, these dendrimers with appropriately modified surfaces are safe, can deliver large plasmids to the brain, and can overcome the cargo size limitations associated with viral vectors. The biocompatibility of this dendritic nanomolecule and the ability to finely tune its surface chemistry provides a gene delivery system that could facilitate future in vivo cellular reprograming and other gene therapies.

Keywords: mixed-surface polyamidoamine dendrimers; gene delivery; glial cells; large plasmid; Sox2

1. Introduction Effective delivery of nucleic acids, such as clustered regularly interspaced short palindromic repeat associated proteins (CRISPR/Cas9), transcription activator-like effectors (TALEs), zinc finger sequences (ZFNs), and small interfering RNA (siRNA), into cells is a critical requirement for cell reprogramming, gene therapy, and vaccine development. In certain cases, only RNA or DNA/plasmids need to be delivered into target cells, while in other cases, concurrent delivery of both DNA and RNA is necessary [1–4]. Nucleic acids are highly polar compounds with numerous negative charges that restrict

Pharmaceutics 2020, 12, 619; doi:10.3390/pharmaceutics12070619 www.mdpi.com/journal/pharmaceutics Pharmaceutics 2020, 12, 619 2 of 15 their entry into membranes with anionic surfaces. In addition, nucleic acids are readily degraded by nucleases that are ubiquitous in the body. Delivery systems capable of protecting nucleic acids against enzymatic destruction and that facilitate their transport into cells are required for gene-based therapy. For in vitro purposes, the delivery system should be able to bind and package nucleic acid cargo to form a complex, which should subsequently bind to the plasma membrane and become internalized, typically by endocytosis. Once inside the cell, the complex should be released from the endosome and the nucleic acid should be expressed [1]. Delivery to an intact organism also requires that the complex is stable in the bloodstream and extracellular space before being taken up by the target cells. Several approaches were reported for the delivery of nucleic acid cargo, each with specific advantages and limitations. Notable examples include viral systems, liposomes, and cationic polymers. Viral systems (such as retrovirus, lentivirus, and adenovirus) are the most frequently used in gene therapy clinical trials due to their high transduction efficiency and prolonged gene expression [1]. The major drawbacks of using viral delivery systems include the immunogenicity of viruses, their link to human diseases, limited capacity of cargo size, and complexity of production [5–10]. Nonviral methods for gene delivery include physical methods, such as the use of voltage or energy to drive DNA into cells or chemicals, such as liposomes and cationic lipids, protein conjugates, linear polymers, dendrimers, or hybrids [11,12]. Compared to viruses, chemical vectors are much simpler systems that are easier to manipulate for the production of safe and reliable pharmaceutical products with batch-to-batch reproducibility. Cationic lipids and liposomes are examples of nonviral delivery systems that are widely used with isolated cells. However, reproducible liposome preparation is challenging and formulations of liposomes with nucleic acids are highly variable and depend on several factors, such as lipid composition, nature of the solvent, and experimental conditions. The physical stability of liposomes may also be compromised by aggregation and coalescence [13–15]. An exception is the class of nanomolecules known as dendrimers. Synthesis of dendrimers is accomplished through a highly controlled process that leads to the formation of precise, branched nanomolecules that form sharp bands on electrophoresis gels similar to proteins [16]. The molecular weight approximately doubles with generation number (G). A polyamidoamine (PAMAM) dendrimer contains three major regions, namely, a central core, a dendritic or branched interior region, and surface functional groups. In addition to amines, PAMAM dendrimers with surfaces made entirely of other functional groups, such as hydroxyls or carboxyls, are also commercially available. The interior region of a PAMAM dendrimer consists of tertiary amines at the branch points and amide linkages in the branches. Since nucleic acids are anionic, binding and packaging require dendrimers with surface amine groups (pK~9) that are cationic, at physiological pH of ~7. Higher generation amine-surface dendrimers ( G4) are more effective in packaging larger quantities of nucleic acid and ≥ bigger plasmids than lower generations ( G3). ≤ One of the most widely studied dendrimers for delivery of nucleic acids in medical applications is the amine-surface PAMAM dendrimer [17–25]. While the surface amines play a pivotal role in DNA packaging and cellular uptake, the tertiary amines located within the interior of the dendrimer play an important role as a buffer to help the dendrimer and its bound cargo escape from the endosome after internalization by the cell. For example, a G4 amine-terminated dendrimer is essentially spherical, with 64 amines on its surface and 62 tertiary amines buried within the globular nanomolecule. Although the G4 amine-terminated dendrimer is highly effective in packaging and delivering nucleic acids into cells, it is also more toxic than the lower generation dendrimers due to the high positive charge density from the 64 primary amines on its surface. Therefore, to circumvent this issue, a nanomolecule with a well-defined and reproducible number of surface amines was designed. The current strategy of reducing dendrimer toxicity involves decreasing the number of surface amines by attaching various chemicals, such as polyethylene glycol (PEG), cyclodextrins, glucocorticoids, methyl groups, amino acids, and several others [19–25]. This strategy requires additional steps of synthesis and purification, complex protection/deprotection steps, is difficult to control, and yields heterogeneous mixtures of dendrimers. The end result is a lack of reproducibility between different batches, a serious Pharmaceutics 2020, 12, 3 of 15 Pharmaceutics 2020, 12, 619 3 of 15 control, and yields heterogeneous mixtures of dendrimers. The end result is a lack of reproducibility between different batches, a serious obstacle in potential clinical trials [26]. We used a much simpler deobstacle novo inmethod, potential and clinical a well-established trials [26]. We PAMAM used a much dendrimer simpler synthesis de novo method, scheme andbased a well-established on Esfand and TomaliaPAMAM [27], dendrimer to reproducibly synthesis prepare scheme a based G4 mixed-surface on Esfand and dendrimer Tomalia [27 (G4-90/10),], to reproducibly as mentioned prepare in aour G4 previousmixed-surface publication dendrimer [28]. (G4-90Briefly,/10), we asstarted mentioned from the in our core previous and synthesized publication each [28 ].generation Briefly, we up started until afrom G3 theester, core which and synthesizedwas then converted each generation into the upfinal until product a G3 ester, (90% which hydroxyl was groups, then converted 10% amine into groups).the final This product G4-90/10 (90% demonstrated hydroxyl groups, about 10% 58 aminehydroxyl groups). groups This (90%) G4-90 and/10 6 demonstratedamines (10%) on about its surface58 hydroxyl (Figure groups 1). (90%) and 6 amines (10%) on its surface (Figure1).

FigureFigure 1. 1. RepresentationRepresentation of the of theG4-90/10 G4-90 with/10 with 90% 90%surface surface hydroxyl hydroxyl groups groups and 10% and surface 10% surfaceamine groups.amine groups.

OurOur long-term goalgoal is is to to treat treat a variety a variety of diseases of diseases through through cellular cellular reprograming reprograming [29–36 ].[29–36]. A specific A specificexample example is the conversion is the conversion of glial cellsof glial into cell neuroblastss into neuroblasts by introducing by introducing the human the SOX2human (hSOX2) SOX2 (hSOX2)gene [36 ].gene To that [36]. end, To wethat demonstrated, end, we demonstrated, as proof-of-principle, as proof-of-principle, that G4-90 /that10 successfully G4-90/10 successfully introduced introducednucleic acids nucleic into cellsacidsin into vitro cellsand in vitro could and deliver could a deliver large plasmid, a large plasmid, 8.4 kb, containing8.4 kb, containing hSOX2 hSOX2 with a withcitrine a reportercitrine reporter gene, into gene, glial into cells glialin vivo cellsvia in intracranial vivo via intracranial injection of theinjection dendrimer–DNA of the dendrimer–/plasmid DNA/plasmidcomplex (dendriplex) complex in (dendriplex) healthy Sprague in healthy Dawley Sprague rats. Dawley rats. 2. Materials and Methods 2. Materials and Methods 2.1. Synthesis of G4-90/10 and Fluorescent Conjugates 2.1. Synthesis of G4-90/10 and Fluorescent Conjugates G4-90/10 was synthesized as previously described [28]. The dendrimers were labeled with fluoresceinG4-90/10 isothiocyanate was synthesized (FITC; as Sigmapreviously Aldrich, descri St.bed Louis, [28]. MO, The USA; dendrimers G4-90/10-FITC) were labeled or Cyanine with fluorescein5.5 (Cy5.5; isothiocyanate Lumiprobe, Hunt(FITC; Valley, Sigma Aldrich, MD, USA; St. Louis, G4-90 /MO,10-Cy5.5) USA; G4-90/10-FITC) as previously describedor Cyanine [ 285.5]. (Cy5.5;Briefly, Lumiprobe, the conjugates Hunt were Valley, purified MD, USA; by dialysis G4-90/10-Cy5.5) using 3 KDa as previously molecular described weight cut-o[28]. ffBriefly,(MWCO) the conjugatesmembranes were (Spectrum purified Labs, by dialysis Rancho using Dominguez, 3 KDa CA,molecular USA) againstweight threecut-off changes (MWCO) of0.9% membranes sodium (Spectrumchloride, followed Labs, Rancho by deionized Dominguez, water overnight,CA, USA)after against which three they changes were lyophilized. of 0.9% sodium The dendrimers chloride, followedwere resuspended by deionized at the water desired overnight, concentration after which in phosphate they were buff lyophilized.ered saline (PBS; The pHdendrimers 7.4) or Hank’s were resuspendedbalanced saline at solutionthe desired (HBSS; concentration Gibco, Waltham, in phos MA,phate USA) buffered for both salinein vitro (PBS;and inpH vivo 7.4)applications. or Hank’s balanced saline solution (HBSS; Gibco, Waltham, MA, USA) for both in vitro and in vivo applications. 2.2. Characterization of G4-90/10 and Conjugates 2.2. Characterization of G4-90/10 and Conjugates Dendrimers and their conjugates were analyzed by acidic native polyacrylamide gel electrophoresisDendrimers [ 16and,37 ]their and isoelectricconjugates focusing were analyzed (IEF) [38]. by Electrophoresis acidic native waspolyacrylamide performed using gel 13 electrophoresisa 10% native gel. [16,37] The gel and was isoelectric run at 100 focusing V for 3.5 (IEF) h at [38]. 4 ◦C. Electrophoresis Dendrimers were was also performed characterized using bya 10%C nativeNMR (usinggel. The Varian gel was Mercury run at 300100 NMRV for spectrometer:3.5 h at 4 °C. Dendrimers proton decoupled were also13C characterized NMR spectrum, by 13CC NMR NMR (using{1H} using Varian a 75 Mercury MHz field; 300 NMR modality spectrometer: of acquisition: proton relaxation decoupled delay, 13C NMR 1.000 spectrum, s; acquisition 13C NMR time, {1H} 1.8 s; using a 75 MHz field; modality of acquisition: relaxation delay, 1.000 s; acquisition time, 1.8 s; number

Pharmaceutics 2020, 12, 619 4 of 15

number of scans, 2000; samples dissolved in D2O) and underwent reverse-phase high-performance liquid chromatography (RP-HPLC; Hitachi HPLC system, Tokyo, Japan) using a C18 column (Varian Microsorb-MV; 250 4.6 mm, 300 A) attached to a MetaGuard 4.6 mm Microsorb 300 A × 5 µ C18 guard column. Mobile phases A and B represented 0.1% aqueous trifluoroacetic acid (TFA) and 0.085% TFA in acetonitrile, respectively. A linear gradient elution of 5–90% B/15 min and a flow rate of 1 mL/min were used. The percentages of surface amines were determined by 2,4,6-trinitrobenzene sulfonic acid assay (TS-28997 Thermo Fisher Scientific, Waltham, MA, USA), using glycine as the standard to prepare the calibration curve.

2.3. Dendrimer Degradation Metabolism of G4-90/10 by various enzymes and physiological chemicals was assessed using acidic native polyacrylamide gel electrophoresis (PAGE) and RP-HPLC.

2.4. In Vitro Introduction of G4-90/10 into HEK293 Cells Human embryonic kidney cells (HEK293; Cell Biolabs, San Diego, CA, USA) were cultured and a stable cell line was maintained in Dulbecco’s modiﬁed eagle’s medium (DMEM; Gibco, Waltham, MA, USA) with 10% fetal bovine serum (FBS; Gibco) and 1% penicillin–streptomycin (P/S; Gibco). We plated 10,000 cells in a 96-well plate containing 0.1 mL media per well. The dendrimers were added to the HEK293 cells at 4 mg/mL (ﬁnal concentration per well) and incubated for 30 min as previously described [28], after which the existing media were removed and fresh media were added. The nuclei of the cells were stained with Hoechst 33,342 (Sigma Aldrich, Saint Louis, MO, USA). Following incubation, the media were replaced with fresh media and the cells were visualized using an Observer Inverted Microscope (Zeiss, Oberkochen, Germany).

2.5. Toxicity of Dendrimer in HEK293 Cells G4-90/10 was added to the cells as described above and incubated for 15 h. Toxicity was determined using the MTT assay [3(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium bromide]. MTT (Sigma Aldrich, Saint Louis, MO, USA) was added to a ﬁnal concentration of 12 mM and the cells were incubated at 37 ◦C for 4 h. Sodium dodecyl sulfate (SDS) was then added and the cells were incubated in the dark at room temperature for 2 h to stop the MTT reaction. When formazan (purple crystals) were observed, absorbance at 570 nm of each well was determined using a SpectraMax M2 (Molecular Devices, San Jose, CA, USA).

2.6. In Vitro Introduction of Reporter Plasmid Dendriplex Reporter plasmids (RPs) were a gift from Dr. Kyle Fink (University of California, Davis, CA, USA). RP1 was 6 kb in size and RP2 was 10 kb in size. Both plasmids contained the mCherry reporter gene expressed under a constitutive promoter. The plasmids were purified from bacteria using Qiagen miniprep kits (Hilden, Germany). The extracted plasmids were quantified using Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and stored at 20 C until − ◦ further use. Dendriplexes (RP1 dendriplex and RP2 dendriplex) were formed by the reaction of G4-90/10-FITC (10 mg/mL in PBS) with RP1 or RP2 in the dark at room temperature for an hour. Dendriplexe size was determined using dynamic light scattering with a Protein Solutions Dyna Pro (Wyatt Technology Corp, Santa Barbara, CA, USA). RP1 and RP2 dendriplexes were incubated with HEK293. Final concentrations of dendrimer and DNA per well were 820 µg/mL and 1.3 µg DNA/mL, respectively, at a ratio of dendrimer amines/DNA phosphate (N/P) of 100:1. The cells were periodically analyzed until the mCherry reporter gene expression was observed using an Observer Inverted Microscope (Zeiss, Oberkochen, Germany). Cell viability was also determined using the MTT assay as described above. Cell transfections with Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) were used as a positive control. Pharmaceutics 2020, 12, 619 5 of 15

2.7. In Vivo Introduction of hSOX2 Plasmid Dendriplex Three 20-week-old Sprague Dawley rats (Charles River Laboratories, Wilmington, MA, USA), were used in this study. All procedures associated with animals followed the guidelines of the Institutional Animal Care and Use Committee (IACUC, Memphis, TN, USA) of Central Michigan University (Mt Pleasant, MI, USA; protocol #18–23 approved on 16 August 2018 and #18–07 approved on 24 May 2018). All rats were housed in clear polycarbonate cages at 22 ◦C with a 12 h light/12 h dark cycle. The animals were given access to food and water ad libitum. The G4-90/10-Cy5.5 dendrimers were complexed with the hSOX2 plasmid (8.4kb; a gift from Michel Sadelain, Addgene plasmid #23242; Addgene, Cambridge, MA, USA) containing the citrine reporter gene at an N/P ratio of 100:1. Delivery of hSOX2 plasmid (8.4 kb) complexed with G4-90/10-Cy5.5 was by intracranial injection into the striata of healthy rats. A 3 µL injection was delivered at the appropriate coordinates (anterior–posterior or A/P, medial–lateral or M/L, and dorsal–ventral or D/V) using a Hamilton syringe (Hamilton, Reno, NV, USA). The coordinates were A/P: 0.30 mm; M/L: − 0.40 mm; D/V: 0.55 mM; and D/V: 0.35 mM. The injection speed was 0.2 µL/min. After each − − − injection, the needle was left in place for 5 min to allow diffusion and then raised slowly. The control rat received the HBSS vehicle. The animals were allowed to recover in their recovery cages. Body temperature was maintained at 37 ◦C using heating pads. The rats were transcardially perfused 72 h after injection with 0.1 M PBS (Sigma Aldrich, Saint Louis, MO, USA), followed by 4% paraformaldehyde (PFA; Sigma Aldrich, Saint Louis, MO, USA) to fix the tissues. The brains were removed and fixed in 4% PFA overnight at 4 ◦C and then transferred to 30% sucrose solution at 4 ◦C until they sank (~2–3 days). They were subsequently frozen using 2-methylbutane (Sigma Aldrich, Saint Louis, MO, USA), packed in dry ice, and stored at 80 C until − ◦ they were sectioned. The brains were sectioned coronally into 30 µm sections using a cryostat set at 20 C (Vibratome UltraPro 5000, St. Louis, MO, USA). Glial cells in the brain tissue were visualized − ◦ by staining with primary antibody against glial fibrillary acidic protein (GFAP) with a prior antigen retrieval step (1 M HCL, pH 0.9). The tissue section was washed 3 10 min in 0.01 M PBS and then × placed in blocking buffer (10% normal goat serum in 0.1% Triton X-100 in 0.01 M PBS) for 1 h at room temperature. The primary antibody against GFAP (AVES, Tigard, OR, USA) was diluted to 1:5000 in 0.1% Triton X 100 in 0.01 M PBS. Primary antibody was applied to the tissue section and incubated − overnight at 4 ◦C. The tissue section was then washed and reacted with labeled secondary antibody (Alexa Fluor 594 goat anti-chicken Ig at 1:500 dilution) for 1 h at room temperature. Cell nuclei were stained with Hoechst 33,342 (Sigma Aldrich, Saint Louis, MO, USA) at a 1:1000 dilution.

3. Results

3.1. Synthesis and Characterization of G4-90/10 We developed a much simpler de novo method for the synthesis of G4-90/10, as previously mentioned in our manuscript [28], based on the original, well-established PAMAM dendrimer synthesis scheme used for synthesis of pure-surface dendrimers [27] (Figure2). An intermediate G3.5-ester was synthesized as previously reported and subsequently reacted with excess ethanolamine (90 equivalents/ester) and ethylenediamine (10 equivalents/ester) to yield the G4-90/10, with a surface composition of 10% amines and 90% hydroxyl groups. The 13C NMR spectrum of the G4-90/10 is shown in Figure3. The amide carbon peaks are at 175 ppm and the rest of the characteristic peaks of G4 PAMAM dendrimer at 32.5, 32.7, 36.6, 41.5, 48.9, and 51.1 ppm are clearly visible. The peaks at around 39.7 and 49.8 ppm (arrow) represent the carbons attached to the surface amine and hydroxyl groups, respectively. Pharmaceutics 2020, 12, 6 of 15

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Figure 2. Synthesis of G4-90/10.

The 13C NMR spectrum of the G4-90/10 is shown in Figure 3. The amide carbon peaks are at 175 ppm and the rest of the characteristic peaks of G4 PAMAM dendrimer at 32.5, 32.7, 36.6, 41.5, 48.9, and 51.1 ppm are clearly visible. The peaks at around 39.7 and 49.8 ppm (arrow) represent the carbons attached to the surface amine and hydroxyl groups, respectively. Figure 2.2. Synthesis of G4-90G4-90/10./10.

Figure 3. 13CC NMR NMR spectrum spectrum of of G4-90/10. G4-90/10.

A spectrophotometric amine assay with 2,4,6-trinitrobenzene sulfonic acid was performed on three batches of G4-90/10 with 10% and 30% surface amines. The averages consistently showed 83% of

Figure 3. 13C NMR spectrum of G4-90/10.

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PharmaceuticsA spectrophotometric2020, 12, 619 amine assay with 2,4,6-trinitrobenzene sulfonic acid was performed7 of on 15 three batches of G4-90/10 with 10% and 30% surface amines. The averages consistently showed 83% of the theoretical values (e.g., 8.3% instead of 10% amines and 25% instead of 30% amines). For the the10% theoretical surface amines, values this (e.g., resulted 8.3% instead in 5.4 amines of 10% aminesinstead andof 6.4 25% amines instead per of dendrimer. 30% amines). For the 10% surfacePAGE amines, is often this used resulted to establish in 5.4 amines the purity instead of ma ofcromolecules 6.4 amines per such dendrimer. as proteins, and is applicable for characterizationPAGE is often used of precise, to establish water-soluble the purity ofsynthetic macromolecules polymers such such as proteins,as PAMAM and isdendrimers applicable for[16,37]. characterization The purity ofof precise, the G4-90/10 water-soluble was similar synthetic to polymersthe pure-surface such as PAMAMdendrimer dendrimers (G4 amine), [16, 37as]. Thedemonstrated purity of the by G4-90the sharp/10 was band similar observed to the on pure-surface the acidic native. dendrimer (G4 amine), as demonstrated by the sharpAs expected band observed from the on PAGE the acidic gel (Figure native. 4, lane 3), dendrimers migrated based on the density of surfaceAs expectedamines. The from pure-surface the PAGE gel amine (Figure dendrimer4, lane 3), dendrimers(100% surface migrated amines) based showed on the the density fastest of surfacemigration amines. (lane The2) followed pure-surface by the amine dendrimer dendrimer with (100% 30% surface amines)amines (lane showed 1), the10% fastest surface migration amines (lane 2)3), followedand 0% surface by the dendrimeramines (pure with hydroxyl 30% surface surface; amines lane (lane 4). It 1),should 10% surfacebe noted amines that the (lane pure 3), andhydroxyl 0% surface surface amines dendrimer (pure (lane hydroxyl 4) also surface;migrated lane in the 4). gel It shoulddue to its be interior noted that tertiary the pureamines, hydroxyl which surfaceare protonated dendrimer under (lane the 4)acidic also conditions migrated inused the for gel electrophoresis. due to its interior The tertiaryisoelectric amines, point (pI) which values are protonatedof different batches under the of the acidic G4-90/10 conditions were useddetermined for electrophoresis. using IEF, as Thepreviously isoelectric reported point [38], (pI) and values were of difoundﬀerent to be batches around of 9.5–10, the G4-90 making/10 were the determineddendrimer a using basic IEF,“protein as previously mimic” (data reported not shown). [38], and were found to be around 9.5–10, making the dendrimer a basic “protein mimic” (data not shown).

[+]

[-]

Figure 4. Acidic native PAGE of the nanomolecule. Lane 1 is the mixed-surface G4 dendrimer with Figure 4. Acidic native PAGE of the nanomolecule. Lane 1 is the mixed-surface G4 dendrimer with 30% surface amines and 70% surface hydroxyl groups, lane 2 is the G4 pure-amine surface dendrimer, 30% surface amines and 70% surface hydroxyl groups, lane 2 is the G4 pure-amine surface dendrimer, lane 3 is the mixed-surface G4 dendrimer with 10% surface amines and 90% surface hydroxyl groups, lane 3 is the mixed-surface G4 dendrimer with 10% surface amines and 90% surface hydroxyl groups, and lane 4 is the G4 pure hydroxyl surface dendrimer. All dendrimers are at 5 µg/lane. and lane 4 is the G4 pure hydroxyl surface dendrimer. All dendrimers are at 5 µg/lane. 3.2. Degradation of Nanomolecules 3.2. Degradation of Nanomolecules We studied the degradation of G4-90/10 using a variety of mammalian enzymes, including lipase, trypsin,We papainstudied and the lysozyme.degradation There of G4-90/10 was no significant using a variety breakdown of mammalian of the dendrimer enzymes, according including to thelipase, enzymes trypsin, studied papain or ratand serum, lysozyme. even There after 24was h incubation no significant at 37 breakdown◦C (data not of shown). the dendrimer We also lookedaccording at the to the effect enzymes of biologically studied relevant or rat serum, chemicals even such after as 24 hypochlorous h incubation acidat 37 (HOCl) °C (data and not hydrogen shown). peroxideWe also looked (H2O2 ),at whichthe effect are of produced biologically by human relevant cells, chemicals especially such phagocytic as hypochlorous cells such acid as (HOCl) neutrophils and andhydrogen macrophages. peroxide (H RP-HPLC2O2), which clearly are produced showed the by diminishedhuman cells, dendrimer especially peakphagocytic at 7.5 mincells andsuch the as appearanceneutrophils ofand new macrophages. peaks, suggesting RP-HPLC breakdown clearly show of theed dendrimer the diminished by 16 mMdendrimer and 32 mMpeak Hat2 O7.52 after min 24and h incubationthe appearance at 37 ◦ofC new (Figure peaks,5A). Thesuggesting native PAGE breakdown showed of that the Hdendrimer2O2 as low by as 16 5 mM mM alone, and 32 as wellmM 2+ asH2O the2 after Fenton 24 h reaction incubation (Fe at/H 372O °C2), (Figure readily 5A). degraded The native G4-90 PAGE/10 (Figure showed5B). that These H2O concentrations2 as low as 5 mM of 2+ Halone,2O2 occuras well in theas the body Fenton as the reaction Km for human (Fe /H red2O2), blood readily corpuscles degraded (RBC) G4-90/10 catalase, (Figure the enzyme 5B). These that breaksconcentrations down H 2ofO 2Hinto2O2 wateroccur andin the oxygen, body whichas the wasKm for reported huma ton red be much blood higher corpuscles at around (RBC) 80 mMcatalase, [39]. HOClthe enzyme (final that concentration breaks down 4%, Hv/2vObleach)2 into water did notand break oxygen, down which the was dendrimer reported under to be these much conditions higher at (dataaround not 80 shown). mM [39]. HOCl (final concentration 4%, v/v bleach) did not break down the dendrimer under these conditions (data not shown).

Pharmaceutics 2020, 12, 8 of 15 Pharmaceutics 2020, 12, 8 of 15 Pharmaceutics 2020, 12, 619 8 of 15

FigureFigure 5. 5. Degradation Degradation of of of G4-90/10 G4-90/10 G4-90/10 as as shown shown by by RP-HPLC RP-HPLC ( A (A) )and) and and acidic acidic native native native PAGE PAGE PAGE ( B( B).). The The top top HPLC trace is the unreacted G4-90/10, the middle trace is the G4-90/10 reacted with 16 mM H2O2, and HPLCHPLC trace trace is isthe the unreacted unreacted G4-90/10, G4-90/10, the the middl middlee trace trace is the is G4-90/10 the G4-90 reacted/10 reacted with with16 mM 16 H mM2O2, H and2O2 , the bottom trace is the dendrimer reacted with 32 mM H2O2.The dendrimer concentration in all cases theand bottom the bottom trace is trace the isdend therimer dendrimer reacted reacted with 32 with mM 32 H mM2O2.The H2O dendrimer2.The dendrimer concentration concentration in all cases in all was 0.07 mM. In the gel, the dendrimer (0.07 mM) was incubated with 80 mM Fe2+/29 mM H2O2 (lane wascases 0.07 was mM. 0.07 In mM. the gel, In the the gel, dendrimer the dendrimer (0.07 mM) (0.07 was mM) incubated was incubated with 80 with mM 80 Fe2+/29 mM Fe2 mM+/29 H mM2O2 (lane H2O 2 1), 50 mM H2O2 (lane 2), 5 mM H2O2 (lane 4), or 500 mM H2O2 (lane 5) in phosphate buffered saline 1),(lane 50 mM 1), 50 H mM2O2 (lane H2O 2),2 (lane 5 mM 2), H 52 mMO2 (lane H2O 4),2 (lane or 500 4), mM or 500 H2 mMO2 (lane H2O 5)2 (lanein phosphate 5) in phosphate buffered bu salineffered (PBS) for 24 h at 37 °C. Lanes 3 and 6 are the dendrimer controls (0.07 mM). (PBS)saline for (PBS) 24 h forat 37 24 °C. h at Lanes 37 ◦C. 3 Lanesand 6 3ar ande the 6 dendrimer are the dendrimer controls controls (0.07 mM). (0.07 mM). 3.3. Toxicity of the Nanomolecule 3.3.3.3. Toxicity Toxicity of of the the Nanomolecule Nanomolecule As expected, the MTT assays showed that the toxicity of G4-90/10 to HEK293 cells in vitro was AsAs expected, expected, the the MTT MTT assays assays showed showed that that the the toxi toxicitycity of of G4-90/10 G4-90/10 to to HEK293 HEK293 cells cells in in vitro vitro was was dramatically decreased compared to the pure 100% surface amine G4 dendrimer. Cells were treated dramaticallydramatically decreased decreased compared compared to to the the pure pure 100% 100% surface surface amine amine G4 G4 dendrimer. dendrimer. Cells Cells were were treated treated with relatively high concentrations of the two dendrimers (4 mg/mL final concentration or 280 µM per withwith relatively relatively high high concentrations concentrations of of the the two two dendrimers dendrimers (4 (4 mg/mL mg/mL final final concentration concentration or or 280 280 µM µM well). Pure-surface amine-treated cells showed 98.5% cell death compared to the control (untreated) perper well).well). Pure-surfacePure-surface amine-treatedamine-treated cellscells showshoweded 98.5%98.5% cellcell deathdeath comparedcompared toto thethe controlcontrol cells. Cells treated with G4-90/10 at the same concentration exhibited only 26.9% death compared to (untreated)(untreated) cells. cells. Cells Cells treated treated with with G4-90/10 G4-90/10 at at the the same same concentration concentration exhibited exhibited only only 26.9% 26.9% death death the untreated cells. Morphological differences were also observed between cells treated with the two comparedcompared to to the the untreated untreated cells. cells. Morphological Morphological diff differenceserences were were also also observed observed between between cells cells treated treated dendrimers. Cells treated with pure-surface amine dendrimer were shrunken and detached from the withwith the the two two dendrimers. dendrimers. Cells Cells treated treated with with pure-surface pure-surface amine amine dendrimer dendrimer were were shrunken shrunken and and culture plate, whereas the cells treated with G4-90/10 were attached and maintained a morphology detacheddetached fromfrom thethe cultureculture plate,plate, whereaswhereas thethe cecellslls treatedtreated withwith G4-90/10G4-90/10 werewere attachedattached andand similar to that of the control cells after 15 h of dendrimer exposure (Figure6). Similar toxicity profiles maintainedmaintained a amorphology morphology similar similar to to that that of of the the control control cells cells after after 15 15 h h of of dendrimer dendrimer exposure exposure (Figure (Figure were also obtained with neurons (data not shown). 6).6). Similar Similar toxicity toxicity profiles profiles were were also also obtained obtained with with neurons neurons (data (data not not shown). shown).

FigureFigure 6. 6. HEK293 HEK293HEK293 cells cells cells treated treated treated with with with pure-surface pure-surface pure-surface G4 G4 100% 100% 100% surface surface surface amine amine amine dendrimer dendrimer dendrimer (A ( (AA),), ),G4-90/10 G4-90/10 G4-90/10 (B ( (B), ),), μ andand untreated untreated control control cells cellscells (C ((C).).). Scale ScaleScale bar barbar = = =100 100 μ m.µm.

Pharmaceutics 2020, 12, 619 99 of of 15 15

3.4. Dendriplex Formation 3.4. Dendriplex Formation Agarose gel electrophoresis was used to ensure complete complex formation of the Agarose gel electrophoresis was used to ensure complete complex formation of the DNA/plasmid. DNA/plasmid. These studies showed that N/P ratios of 1:1, 10:1, and 100:1 were able to form These studies showed that N/P ratios of 1:1, 10:1, and 100:1 were able to form complexes with the complexes with the plasmid. However, the 100:1 ratio was found to form complexes with all of the plasmid. However, the 100:1 ratio was found to form complexes with all of the plasmid, compared to plasmid, compared to the 1:1 and 10:1 ratios which showed only faint plasmid bands just below the the 1:1 and 10:1 ratios which showed only faint plasmid bands just below the well, indicating negligible well, indicating negligible amounts of free plasmid available; this was absent in well 3, corresponding amounts of free plasmid available; this was absent in well 3, corresponding to the 100:1 complex to the 100:1 complex (Figure 7). Dynamic light scattering showed that the average hydrodynamic (Figure7). Dynamic light scattering showed that the average hydrodynamic radius of complexes radius of complexes formed between the 10 kb RP and G4-90/10 at N/P of 100:1 was ~135 nm. formed between the 10 kb RP and G4-90/10 at N/P of 100:1 was ~135 nm.

Figure 7. AgaroseAgarose gel electrophoresis showsshows thethe didifferencfferencee in in migration migration of of the the RP2 RP2 when when complexed complexed at atdi ffdifferenterent N /N/PP ratios, ratios, such such as 100:1as 100:1 (lane (lane 3), 3), 10:1 10: (Lane1 (Lane 4), and4), and 1:1 1:1 (lane (lane 5). Lanes5). Lanes 1 and 1 and 2 represent 2 represent the the100 100 bp ladderbp ladder and and free free plasmid plasmid (arrow), (arrow), respectively. respectively. The The absence absence of free of free plasmid plasmid bands bands in lanes in lanes 3, 4, 3,and 4, and 5 shows 5 shows that that the RP2the RP is complexed2 is complexed with with G4-90 G4-90/10./10. 3.5. In Vitro Uptake of G4-90/10 3.5. In Vitro Uptake of G4-90/10 Fluorescence microscopy showed that the G4-90/10-Cy5.5 was successfully taken up by HEK293 Fluorescence microscopy showed that the G4-90/10-Cy5.5 was successfully taken up by HEK293 cells following incubation for 30 min at a final concentration of 4 mg/mL, as shown by the localization cells following incubation for 30 min at a final concentration of 4 mg/mL, as shown by the localization of Cy5.5 in cells; Hoechst staining shows the cell nuclei (Figure8). of Cy5.5Pharmaceutics in cells; 2020 Hoechst, 12, staining shows the cell nuclei (Figure 8). 10 of 15

(A) (B) (C) Figure 8. Uptake and retention of G4-90/10-Cy5.5 by HEK293 cells. Cell nuclei labeled with Hoechst Figure33,342 (arrow)8. Uptake are and shown retention in (A of), G4-90/10-Cy5.5 (B) shows the uptakeby HEK of293 G4-90 cells./10-Cy5.5 Cell nuclei dendrimers labeled with by HEK293Hoechst 33,342cells (arrow), (arrow) and are (shownC) is the in merged(A), (B) imagesshows the of theuptake Hoechst of G4-90/10-Cy5.5 and G4-90/10-Cy5.5 dendrimers staining, by HEK293 showing cells that (arrow),G4-90/10-Cy5.5 and (C) are is the taken merged up by images the HEK293 of the cells.Hoechst Scale an bard G4-90/10-Cy5.5= 100 µm. staining, showing that G4- 90/10-Cy5.5 are taken up by the HEK293 cells. Scale bar = 100 μm. The image shown in Figure8 reveals that the transfection e ﬃciency (number of cells stained with

Cy5.5The/number image of shown nuclei in stained Figure with 8 reveals Hoechst) that was the ~100%. transfection efficiency (number of cells stained with Cy5.5/numberFigure 8. Uptake of nuclei and retention stained of G4-90/10-Cy5.5with Hoechst) by wasHEK 293~100%. cells. Cell nuclei labeled with Hoechst 33,342 (arrow) are shown in (A), (B) shows the uptake of G4-90/10-Cy5.5 dendrimers by HEK293 cells (arrow), and (C) is the merged images of the Hoechst and G4-90/10-Cy5.5 staining, showing that G4- 90/10-Cy5.5 are taken up by the HEK293 cells. Scale bar = 100 m.

The image shown in Figure 8 reveals that the transfection efficiency (number of cells stained with Cy5.5/number of nuclei stained with Hoechst) was ~100%.

Pharmaceutics 2020, 12, 10 of 15

3.6. In Vitro Introduction of RP1 and RP2 and Toxicity Fluorescence microscopy showed that the G4-90/10 successfully delivered the 6 kb and 10 kb plasmids to HEK293 cells at an N/P ratio of 100:1 and expressed the mCherry reporter gene (Figure Pharmaceutics9). The solid2020 arrows, 12, 619 in panels A and D (Figure 9) show the FITC fluorescence exhibited by10of the 15 dendrimer component of the dendriplex carrying RP1 and RP2, respectively. The solid arrows in 3.6.panels In VitroB and Introduction E show the of mCherry RP1 and fluorescence RP2 and Toxicity establishing expression of the RP1 and RP2 plasmids, respectively. Colocalization of the FITC fluorescence from the dendrimer and the red fluorescence fromFluorescence the mCherry microscopyin the cytoplasm showed revealed thatthe that G4-90 the dendriplexes/10 successfully were delivered taken up theby the 6 kb cells and and 10 the kb plasmidsmCherry toreporter HEK293 gene cells was at anexpressed N/P ratio after of 100:1 14 h incu andbation expressed (solid the arrows; mCherry images reporter C and gene F). (Figure The open9). Thearrow solid in image arrows F in shows panels a Acell and expressing D (Figure the9) show mCherry the FITC protein fluorescence without any exhibited green byfluorescence the dendrimer from componentthe dendrimer, of the indicating dendriplex that carrying the dendrimer RP1 and was RP2, exocytosed. respectively. The The arrowhead solid arrows shows in a panels cell where B and the E showdendriplex the mCherry was inside fluorescence the cell, but establishing before mCherry expression was ofexpressed. the RP1 and The RP2 images plasmids, shown respectively. in Figure 9 Colocalizationindicate that the of theexpression FITC fluorescence efficiency from(number the dendrimerof expressing and mCherry/number the red fluorescence of fromcells thestained mCherry with inG4-90/10-FITC) the cytoplasm was revealed about that 33%. the dendriplexes were taken up by the cells and the mCherry reporter geneComparing was expressed the aftertoxicity 14 of h incubationthe complex (solid formed arrows; with G4 images 100% C pure-surface and F). The amine open arrow dendrimer in image and FG4-90/10, shows a only cell expressing 33% of the the cells mCherry treated proteinwith the without pure-amine any greensurface fluorescence dendriplex from survived the dendrimer, compared indicatingto 97% survival that the with dendrimer the G4-90/10 was exocytosed. dendriplex. The Si arrowheadmilar results shows were a cellobtained where thewith dendriplex the 6 kb wasRP1 insideplasmid the (results cell, but not before shown). mCherry was expressed. The images shown in Figure9 indicate that the expression efficiency (number of expressing mCherry/number of cells stained with G4-90/10-FITC) was about 33%.

FigureFigure 9.9. ImagesImages ((A,)D and) show (D) thatshow the that dendriplexes the dendriplexes formed form withed G4-90 with /10-FITCG4-90/10-FITC and RP1 and are RP1 taken are uptaken by theup by HEK293 the HEK293 cells; images cells; images (B,E) show (B) and mCherry (E) show expression mCherry from expression RP1 and from RP2; RP1 images and (RP2;C,F) (solidimages arrow) (C) and show (F) colocalization(solid arrow) betweenshow colocalization the G4-90/10-FITC between and the the G4-90/10-FITC mCherry. The and open the arrow mCherry. and theThe arrowhead open arrow in and image the F arrowhead show mCherry in image expression F show but mCherry exocytosed expression G4-90/10-FITC but exocytosed and G4-90 G4-90/10-/10-FITC µ deliveryFITC and before G4-90/10-FITC mCherry isdelivery expressed, before respectively mCherry is (Scale expressed, bar: 100 respectivelym). (Scale bar: 100 µm). Comparing the toxicity of the complex formed with G4 100% pure-surface amine dendrimer and 3.7. In Vivo Transfection G4-90/10, only 33% of the cells treated with the pure-amine surface dendriplex survived compared to 97% survivalIntracranial with administration the G4-90/10 dendriplex. of the dendriplexes Similar results showed were colocalization obtained with between the 6 kb hSOX2 RP1 plasmid (Figure (results10B), GFAP not shown). (Figure 10C), and Cy5.5 (Figure 10D), demonstrating successful uptake by the glial cells

3.7. In Vivo Transfection

Intracranial administration of the dendriplexes showed colocalization between hSOX2 (Figure 10B), GFAP (Figure 10C), and Cy5.5 (Figure 10D), demonstrating successful uptake by the glial cells expressing the citrine reporter gene; Hoechst staining was used to identify cells (Figure 10A) (Figure 10). Pharmaceutics 2020, 12, 11 of 15

Pharmaceuticsexpressing2020 the ,citrine12, 619 reporter gene; Hoechst staining was used to identify cells (Figure 10A) (Figure 11 of 15 10).

FigureFigure 10.10. ImageImage (A (A) shows) shows cells cells labeled labeled with withHoechst; Hoechst; image image(B) shows (B) citrine shows expression citrine expression from the from thehSOX2 hSOX2 plasmid; plasmid; image image (C) shows (C) showsglial fibrillary glial fibrillary acidic protein acidic (GFAP) protein expression; (GFAP) image expression; (D) shows image (D) showsfluorescence fluorescence from G4-90/10-Cy5.5; from G4-90/10-Cy5.5; image (E) imageis the merge (E) is between the merge all imag betweenes, confirming all images, that confirming the G4- that the90/10-Cy5.5 G4-90/10-Cy5.5 dendrimers dendrimers successfully successfully delivered the delivered large hSOX2 the plasmid large hSOX2 in vivo and plasmid were successfullyin vivo and were taken up by the glial cells 72 h post-injection (scale bar: 100 µm). successfully taken up by the glial cells 72 h post-injection (scale bar: 100 µm).

4.4. Discussion Discussion OurOur research focus focus is ison onthe thedelivery delivery of small of smallmolecule molecule drugs and drugs macromolecules and macromolecules into the brain into the for the treatment of a variety of diseases. One of our specific goals is to reprogram a few glial cells in brain for the treatment of a variety of diseases. One of our specific goals is to reprogram a few the brain into functional neurons for treatment of neuronal loss in neuropathological conditions, such glial cells in the brain into functional neurons for treatment of neuronal loss in neuropathological as stroke. Reprogramming endogenous cells by inducing expression of one or more transcription conditions, such as stroke. Reprogramming endogenous cells by inducing expression of one or more factors is a promising alternative to transplantation-based therapy. A variety of somatic cells were transcriptionsuccessfully factorsreprogrammed, is a promising including alternative brain toastrocytes transplantation-based into neurons, therapy.heart fibroblasts A variety into of somatic cellscardiomyocytes, were successfully and exocrine reprogrammed, cells of the including pancreas braininto insulin-secreting astrocytes into beta-cells neurons, [29–35]. heart fibroblasts Su and into cardiomyocytes,colleagues reported and that exocrine delivery cells of ofSOX2 the pancreasDNA to less into than insulin-secreting 10% of the astrocytes beta-cells present [29–35 was]. Su and colleaguessufficient to reported reprogram that them delivery into neuroblasts of SOX2 capable DNA toof maturing less than into 10% synapse-forming of the astrocytes neurons present in was sutheffi cientinjured to reprogram adult spinal them cord into of neuroblasts mice treated capable with of valproic maturing acid into [36]. synapse-forming Since this involved neurons in the injuredreprogramming adult spinal only, cord a small of mice percentage treated of with glial valproic cells was acid required [36]. Since for therapeutic this involved effect reprogramming and the only,development a small percentageof a highly ofreproducible, glial cells wasnonviral required delivery for therapeuticsystem with ereducedffect and toxicity the development was more of a highlyimportant reproducible, than its transfection nonviral ability. delivery system with reduced toxicity was more important than its transfectionA spectrophotometric ability. amine assay with 2,4,6-trinitrobenzene sulfonic acid, a sensitive reagent for the determination of amino groups in amino acids and proteins ,was used to quantitate the A spectrophotometric amine assay with 2,4,6-trinitrobenzene sulfonic acid, a sensitive reagent for number of surface amine groups per dendrimer. Glycine was used to prepare a calibration curve. the determination of amino groups in amino acids and proteins, was used to quantitate the number of Mixed-surface PAMAM dendrimers, such as G4-90/10, possess both amines that can be surfaceprotonated amine to groupsgive positive per dendrimer. charges and Glycine hydroxyls was that used can to become prepare deprotonated a calibration to curve. give negative charges.Mixed-surface Given this, PAMAM IEF is a useful dendrimers, technique such to as determine G4-90/10, the possess isoelectric both aminespoints of that mixed-surface can be protonated todendrimers, give positive similar charges to andproteins hydroxyls [38]. thatThe can pI become values deprotonatedof amidoethanol to give surface negative and charges. Giventris(hydroxymethyl)amidomethane this, IEF is a useful technique (TRIS) to determine surface PAMAM the isoelectric dendrimers, points both of mixed-surface consisting of dendrimers,100% similarhydroxyl to proteinsgroups, [38were]. The reported pI values to ofbe amidoethanolaround 9 and surface 7, respectively and tris(hydroxymethyl)amidomethane [38]. The amidoethanol (TRIS)dendrimers surface showed PAMAM a pI dendrimers,> 7 due to their both tertiary consisting amines. of 100%Lower hydroxylpI values groups,of the TRIS were surface reported to bedendrimer around 9are and likely 7, respectivelydue to the presence [38]. Theof some amidoethanol surface carboxyl dendrimers groups, which showed occur a pIas >a result7 due of to their tertiaryester hydrolysis amines. Lowerduring synthesis. pI values The of the pI TRISvalues surface of different dendrimer batches areof the likely G4-90/10 due to were the found presence to be of some surfacearound carboxyl 9–10, making groups, the dendrimer which occur a basic as a “protein result of mimic” ester hydrolysis [28]. Basic duringproteins, synthesis. such as cytochrome The pI values of c (a protein that travels along the inner mitochondrial membrane) and histones (proteins that different batches of the G4-90/10 were found to be around 9–10, making the dendrimer a basic “protein condense DNA), exhibit pI values between 10–11, while some natural antibodies and therapeutic mimic” [28]. Basic proteins, such as cytochrome c (a protein that travels along the inner mitochondrial monoclonal antibodies show pI values as high as 9.5. The basic nature of G4-90/10 facilitates its membrane)binding to anionic and histones cell membranes (proteins as that well condense as DNA. DNA), G4-90/10 exhibit conjugates pI values with betweenfluorescent 10–11, dyes while also some naturalexhibited antibodies sharp protein-like and therapeutic bands on monoclonalacidic native antibodiesgels [28]. show pI values as high as 9.5. The basic nature of G4-90/10 facilitates its binding to anionic cell membranes as well as DNA. G4-90/10 conjugates with fluorescent dyes also exhibited sharp protein-like bands on acidic native gels [28]. Long-term accumulation of chemicals that cannot be degraded within cells often leads to adverse health effects [40]. Thus, an important concern for clinical applications is the biodegradation of dendrimer-based nanomedicines under physiological conditions. PAMAM dendrimers smaller than G5 are readily excreted by the kidneys, while generations higher than G5 accumulate in the liver and Pharmaceutics 2020, 12, 619 12 of 15 other organs [41]. Proteolytic enzymes, such as trypsin, chymotrypsin, and papain, did not degrade G4-90/10. Lipases that hydrolyze esters in lipid particles also did not break down the dendrimer. There was also no significant breakdown of the dendrimer by the enzymes present in rat serum. However, H2O2, which is produced by human cells, especially phagocytic cells such as neutrophils and macrophages, was found to be effective in degrading G4-90/10. Several in vitro and a few in vivo toxicity studies were reported for PAMAM dendrimers [42–47]. Studies on the development of zebrafish embryos showed that surface amine PAMAM dendrimers are more toxic than hydroxyl or carboxyl surface dendrimers [46,47]. Although the in vitro and in vivo toxicity levels of PAMAM dendrimers are not necessarily correlated [42], a common feature reported in most of the previous studies is the significantly higher toxicity associated with amine-surface dendrimers compared to dendrimers with hydroxyl or carboxyl surfaces. The high positive charge density on the surface of amine-surface dendrimers (or other cationic polymers), although beneficial for DNA packaging, is not found in nature and results in complex formation by anionic biomolecules such as serum proteins and membranes, resulting in protein cross-linking and aggregation, blood clotting, hemolysis, membrane damage, oxidative stress, apoptosis, and other deleterious effects [42]. Hence, the need is present for a high-generation dendrimer with minimal surface amines sufficient for complexing with DNA, but with an improved safety profile. As expected, the MTT assay showed that the toxicity of G4-90/10 to HEK293 cells in vitro was significantly reduced compared to cells challenged with pure-surface amine G4 dendrimer. Most previous studies used much lower dendrimer concentrations (<100 µM). For example, a dose as low as 6 µM G4 amine-surface PAMAM dendrimer killed over 80% of mouse macrophage J774A.1 cells [45]. In addition to dramatically reduced cellular toxicity, all animals treated with G4-90/10 showed normal feeding, movement, and other behavioral features for at least one week before sacrifice. The G4-90/10 readily formed dendriplexes with plasmids, even as large as 10 kb. It should be noted that the final concentration of the dendrimers in the dendriplex was 0.82 mg/mL, compared to toxicity studies performed with dendrimers alone which resulted in 4 mg/mL (Figure6). in vitro and in vivo studies showed that the G4-90/10 dendrimer entered cells in spite of a ten-fold reduction in surface amines. In addition, the reduced surface amines did not prevent the dendrimer from forming complexes (dendriplexes) with large DNA plasmids. It is highly possible that the cationic tertiary amines below the dendrimer surface helped to complex the DNA. Most importantly, the dendriplexes were taken up by cells both in vitro and in vivo and the genetic cargo was expressed, suggesting that these surface-modified dendrimers might offer an excellent alternative to viruses for nucleic acid delivery.

5. Conclusions We designed and synthesized a gene delivery nanomolecule with a favorable safety profile that should be applicable for in vivo cell reprogramming. Unlike modification of dendrimer surfaces to reduce or shield cationic amines, we carried out de novo synthesis of the nanomolecule, which yielded a purer product as demonstrated electrophoresis and chromatography. Despite the limited number of surface amines present, the G4 mixed-surface dendrimer was able to complex large plasmids and deliver them to cells in vitro and in vivo, without interfering with their expression. The decreased cationic surface of the dendrimer and its degradation by physiological amounts of hydrogen peroxide increased the safety profile of the nanomolecule, which is important regarding potential use in human clinical applications.

Author Contributions: B.S. conducted the experiments of the study, participated in the design of the study, analyzed the data, and helped draft the manuscript; M.F. conducted the experiments of the study, participated in the design of the study, and analyzed the data; B.C. and K.J. conducted the chemistry-based experiments of the study and analyzed the data; D.S. synthesized the dendrimers; N.M., S.P., A.A., M.A., and A.F. conducted the experiments of the study and analyzed the data; A.S., G.L.D., and J.R. conceived the study, participated in the design and coordination, edited, and wrote the ﬁnal proof of the manuscript. All authors have read and agreed to the published version of the manuscript. Pharmaceutics 2020, 12, 619 13 of 15

Funding: This study was funded by the Neuroscience program, the College of Medicine, the Field Neurosciences Institute, the Department of Chemistry and Biochemistry, and the John G. Kulhavi Professorship in Neuroscience at CMU. Support for part of the project was provided by the American Heart Association (AHA grant #18AIREA33990094). Conﬂicts of Interest: The authors declare no conﬂict of interest.

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