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. Author manuscript; available in PMC: 2026 Sep 29.
Published in final edited form as: Biomed Mater Devices. 2023 Aug 16;2(1):529–540. doi: 10.1007/s44174-023-00122-8

Tuning the pH-Responsive Properties of (2-Diisopropylamino) Ethyl Methacrylate-Based Cationic Nanoparticles: Toward Enhanced Nanoparticle Stability and Cytocompatibility

Aaliyah Shodeinde 1,2, Olivia L Lanier 2,3, Shreya Nallaparaju 2,4, Michael Lam 2,3, Ani Savk 1, Nicholas A Peppas 1,2,3,4,5,6,7
PMCID: PMC13619317  NIHMSID: NIHMS2204430  PMID: 42808097

Abstract

Cationic nanoparticles have been widely investigated for their potent ability to transport therapeutic agents intracellularly. Previous studies in our lab have demonstrated that increasing network hydrophobicity in these polymers induces favorable pH-responsive phase transition and membrane-disruptive behavior for intracellular drug delivery. Building upon this idea, the work presented herein analyzes the physiochemical attributes and cargo delivery efficiency of a cationic nanoparticle system with the introduction with 2-(diisopropylamino)ethyl methacrylate (henceforth designated as DPAEMA), a comonomer heightening the hydrophobic content of the inner particle core. DPAEMA is a cationic, amine-containing monomer often introduced as an ionizable hydrophobic block to nanoparticles due to its high biocompatibility and ability to impart pH-sensitivity with a pKa of around 6.2. DPAEMA bears a hydrophobic nature under pH conditions greater than its pKa from protonation and rapidly transitions into a hydrophobic state under pH conditions lower than 6.2 (acidic) from deprotonation which makes it an ideal candidate for use as a comonomer or as the main functional monomer when synthesizing pH-responsive nanoparticles for use in biological applications. In addition, the use of monomers with tertiary amines as a functional group offers other desired properties to hydrogels and nanostructures, such as antimicrobial behavior and good biocompatibility characteristics. Most studies with DPAEMA have only explored the delivery of hydrophobic, non-charged anticancer agents wherein the release of the payload is aided by diffusion out of the nanocarriers to the target location. Generally, nanoparticles synthesized with DPAEMA in the literature for drug delivery applications have been small (less than 150 nm) with hydrodynamic diameters varying between studies due to the nature of comonomers or differences in the nominal feed ratios of DPAEMA used. Due to the limited number of studies investigating the function of DPAEMA in cationic nanoparticles, it is imperative to attain a deeper comprehension of its solution behavior to optimize the properties of these vectors. In our work presented here we concentrated upon the synthesis of stable cationic DPAEMA nanoparticles with controlled particle nanometric size and stability for drug-release applications. We investigate the following key factors impacting the responsive characteristics of the particle: hydrodynamic size, pKa, volume swelling ratio, and the pH range of particle swelling. Furthermore, a cell proliferation assay was determined to evaluate particle cytocompatibility, membrane disruption, and transfection capabilities. The insights obtained from these studies will facilitate the design of novel drug delivery platforms for intracellular transport by explicating the role of charge and hydrophobicity in the operation of cationic nanoparticle systems.

Keywords: Hydrogels, Protein delivery, Biomaterials, Nanoparticles

Introduction

The application of nanoparticles as novel drug delivery systems has garnered considerable interest for their ability to harness the singular properties of nanomaterials to overcome the limitations of conventional pharmaceutical formulations in terms of active targeting, sustained release, reduced toxicity, and lower production costs [1–3]. Furthermore, recent advancements in nanoparticle synthesis strategies impart unprecedented control over chemical composition as well as functionality and enable the tunability of these polymeric systems (i.e., compositional characteristics of the particle chemistry or the addition of complex architectures and bio-responsive moieties) for a particular end use [4].

Cationic nanoparticles in particular have been widely investigated for their potent ability to transport therapeutic agents intracellularly. Previous studies in our lab have demonstrated that increasing network hydrophobicity in these polymers induces favorable pH-responsive phase transition and membrane-disruptive behavior for intracellular drug delivery [5–7]. Additional work has also reported on the role of hydrophobicity in modulating the critical swelling pH, cytotoxicity, cell uptake, and membrane destabilization properties of cationic particles. Building upon this idea, the work presented herein analyzes the physiochemical attributes and cargo delivery efficiency of a cationic nanoparticle system with the introduction with 2-(diisopropylamino) ethyl methacrylate (henceforth designated as DPAEMA), a comonomer heightening the hydrophobic content of the inner particle core. DPAEMA is a cationic, amine-containing monomer often introduced as an ionizable hydrophobic block to nanoparticles due to its high biocompatibility and ability to impart pH-sensitivity with a pKa of around 6.2 [8, 9]. DPAEMA bears a hydrophobic nature under pH conditions greater than its pKa from protonation and rapidly transitions into a hydrophobic state under pH conditions lower than 6.2 (acidic) from deprotonation which makes it an ideal candidate for use as a comonomer or as the main functional monomer when synthesizing pH-responsive nanoparticles for use in biological applications [10, 11]. In addition, the use of monomers with tertiary amines as a functional group offers other desired properties to hydrogels and nanostructures, such as antimicrobial behavior and good biocompatibility characteristics [12–17].

DPAEMA has been combined with comonomers or 2-hydroxyethyl methacrylate (HEMA) and is frequently copolymerized with either poly(ethylene glycol) monomethacrylate (PEGMA) or poly(glycerol monomethacrylate) (PGMA) for improved colloidal stability and prolonged lifespan in vivo [12, 18]. Atom transfer radical polymerization (ATRP), reversible addition-fragmentation chain transfer (RAFT) polymerization [19], and nano-precipitation are common methods used for the synthesis of responsive polymer networks based on DPAEMA [18, 20].

DPAEMA nanoparticles have garnered significant interest in the field of anticancer drug delivery, as the hydrophobicity and toxicity of the drugs in question have proven to be incompatible with conventional delivery approaches [21]. Nanoparticles utilized for targeted drug delivery in cancer rely on two main phenomena for successful localization: the enhanced permeability and retention (EPR) effect and the more acidic pH of the tumor microenvironment (pH @ 7.2) relative to its surrounding environment (blood pH @ 7.4) [22, 23]. As such, when the pH-sensitive nanoparticles reach the acidic tumor microenvironment, the excess of protons in the region induces the nanoparticles to swell in size [24–27]. The protonation of the amine group in DPAEMA into a soluble quaternary ammonium salt under endosomal pH conditions causes the polymer chains to achieve a hydrophobic to hydrophilic transition which facilitates the dissociation of polymeric micelles or the extension of the polymer chains within crosslinked nanoparticles to control drug release [20, 28].

Most studies with DPAEMA have only explored the delivery of hydrophobic, non-charged anticancer agents wherein the release of the payload is aided by diffusion out of the nanocarriers to the target location. As the EPR effect and acidic tumor microenvironment remain true for most cancer types, applications of the DPAEMA infrastructure to encapsulate treatments for colon cancer [10], breast cancer [18], and ovarian cancer [18] have been studied. Faccia and colleagues have applied a similar concept for the development of therapeutic contact lenses by relying on deviations from normal ocular pH conditions (pH 7.4), which have been observed in diseases, such as in ocular rosacea, to trigger the pH responsiveness [12]. Anti-cancer studies involving DPAEMA nanoparticles rely on physical entrapment of the payload without any drug interactions between the drug and polymer due to the hydrophobic nature of anticancer drugs, like doxorubicin (DOX) and paclitaxel (PTX) [29–32]. This was the case in the loading of dexamethasone 21-disodium phosphate (DXP) into DPAEMA hydrogels utilized for therapeutic contact lenses [12]. The use of physical entrapment is advantageous because it translates to more general applications for other types of drugs, provided the drug is soluble in the loading medium, the drug can be encapsulated into the nanoparticle [33–35].

Generally, nanoparticles synthesized with DPAEMA for drug delivery applications were small with hydrodynamic diameters varying between studies due to the nature of comonomers or differences in the nominal feed ratios of DPAEMA used. The hydrodynamic diameter of the nanoparticles ranged from 42 to 132 nm which is optimal for applications, such as intracellular drug delivery, as these sizes are well above the range of renal clearance (> 5–10 nm) but still small enough to pass into the leaky vasculature environment of tumors (under 200 nm) [5, 35].

Due to the limited number of studies investigating the function of DPAEMA in cationic nanoparticles, it is imperative to attain a deeper comprehension of its solution behavior to optimize the properties of these vectors. In our work presented here we concentrated upon the synthesis of stable cationic DPAEMA nanoparticles with controlled particle nanometric size and stability for drug-release applications. We investigate the following key factors impacting the responsive characteristics of the particle: hydrodynamic size, pKa, volume swelling ratio, and the pH range of particle swelling. Furthermore, a cell proliferation assay was determined to evaluate particle cytocompatibility, membrane disruption, and transfection capabilities. The insights obtained from these studies will facilitate the design of novel drug delivery platforms for intracellular transport by explicating the role of charge and hydrophobicity in the operation of cationic nanoparticle systems. These DPAEMA-based nanoparticles with tunable properties can be used to address a myriad of challenges in the application of cationic nanoparticles for biomedical applications [36, 37].

Materials and Methods

Materials

Monomers 2-(Diisopropylamino)ethyl methacrylate (DPAEMA) (Cat. No. 730971), 2-(diethylamino)ethyl methacrylate (DEAEMA, 99%) (Cat. No. 408980), tert-butyl methacrylate (tBMA) (Cat. No. 463353), 98%), cyclohexyl methacrylate (CHMA) (Cat. No. 408964), bis(2-methacryloyl)oxyethyl disulfide (DSDMA), Poly(ethylene glycol) methyl ether methacrylate solution (PEGMA2k, Mn = 2000) (Cat. No. 457876), myristyl trimethylammonium bromide (MyTAb) (Cat. No. T4762), ethyl α-bromoisobutyrate (EBiB, 98%) (Cat. No. 252271), DMSO-d6 (99.8% D) (Cat. No. 151874), trimethylsilanol (> 97.5% GC), and L-ascorbic acid (AA) (Cat. No. A7506) were purchased from Sigma-Aldrich. Hexyl methacrylate (Cat. No. M0503) and Tris(2-pyrdiylmethyl)amine (Cat. No. T2671) were purchased from TCI America. Brij 30 (Cat. Code 216725000), Brij 35 (Cat. No. 228345000), and copper (II)bromide (99 + %) (Cat. No. 206331000) were purchased from Acros Organics. Tris(2-carboxyethyl)phosphine (TCEP) (Cat. No. 580560) was purchased from EMD Millipore. Tetrahydrofuran, acetone, ethanol, and hydrochloric acid were purchased from Fisher Scientific. Silencer™ Negative Control No. 1 siRNA (Cat. No. AM4635) was purchased from Thermo Fisher Scientific. CellTiter 96 AQueous One Solution Cell Proliferation Assay (Cat. No. PR-G3580) and QuantiFluor® RNA System (Cat. No. E3310) was purchased from Promega. Caco-2, RAW 264.7, and THP-1 cells were obtained from ATCC.

Synthesis and Purification of Cationic Nanogels

The nanoparticles were synthesized using a one-pot oil-in-water emulsion polymerization scheme via an ATRP method based on activators regenerated by electron transfer (henceforth designated as ARGET) that has been previously described in detail [38].

Briefly, the emulsions were prepared by adding all the reagents into a 1-dram glass vial in 2-mL batch sizes and then homogenized for 20 min using a 24-tip microtip sonicator probe. Next, the samples were transferred into an MBraun Glovebox LABmaster 130 under inert nitrogen conditions (O2 < 0.1 ppm; H2O < 0.1 ppm) to initiate the reaction with the addition of ascorbic acid. After 24 h, the reaction was quenched by exposing the samples to air and diluting the concentration to 0.5 N through the addition of 4-N HCl. Unreacted monomer and additional impurities were extracted from the nanoparticles using ionomer collapse, defined as the process of acidifying the samples and then precipitating with a low dielectric constant solvent, like tetrahydrofuran. The nanogels were precipitated through ionomer collapse thrice and retrieved after each cycle via centrifugation at 4000×g for 5 min. Following the final centrifuge, nanoparticles were resuspended in deionized water and dialyzed against deionized water for at least 7 days to remove any trace amounts of tetrahydrofuran.

Determination of Nanogel Composition

Nanoparticles for spectroscopic characterization were lyophilized to obtain the dry, crosslinked polymers. These polymers were characterized via Attenuated Total Reflectance Fourier Transform Infrared (ATR-FTIR) Spectroscopy (Thermo Scientific Nicolet iS10 with a germanium crystal) and 1H Nuclear Magnetic Resonance (NMR) Spectroscopy utilizing a 500-MHz Bruker AVANCE III 500 (Prodigy 500).

For ATR-FTIR measurements, background spectra collections (analyzed using the Omnic 9 software) were obtained before each sample and used for background subtraction over 64 scans. NMR samples were dissolved in DMSO-d6 with 0.03 v/v% trimethylsilanol at 10 mg/mL and degraded for 24 h with the addition of 10-mM TCEP. TCEP is a preferred powerful and irreversible reducing agent utilized in the field by many researchers [39]. Spectra were collected for a total of 64 scans and analyzed on MestReNova 14.2.3 software.

Hydrodyna Mic Size and Electrophoretic Potential

The nanoparticle hydrodynamic diameters and zeta potentials were measured using the dynamic light scattering (DLS) and zeta potential protocols on a Malvern ZetaSizer NanoZS. The nanoparticles were prepped for DLS characterization by diluting the stock solution of nanoparticles 20 times in 1X PBS to give a final 1.5-mL volume. Samples were analyzed at pH 4.5 (acidic) and pH 7.4 (neutral) to evaluate the change in nanoparticle hydrodynamic size as well as the degree of monodispersity of the nanoparticles (measured as a polydispersity index (PDI)) at different pH conditions. Electrophoretic (zeta) potential was measured at a 20X nanoparticle dilution in 5 mM sodium phosphate buffer at similar acidic and neutral pH conditions. All DLS and zeta potential measurements were collected in triplicates utilizing standard operating procedures optimized to the cationic nanoparticles.

Critical Swelling pH

The dynamic swelling behavior of different nanoparticle formulations in response to a change in pH was determined using the MPT-2 multipurpose titrator attachment of the Malvern ZetaSizer NanoZS. DLS measurements were conducted in triplicates between pH 4.0 and 7.0 or 8.0 (depending on the nanoparticle composition) at increments of 0.3-pH units. The nanoparticle stock solution was diluted 10X in 1X PBS for these studies. The automated adjustment of the pH of the samples was accomplished with 0.1-N HCl, 1-N HCl and 1-N NaOH.

Cell Culture of Caco-2 Human Colonic Epithelial Cells and RAW 264.7 Murine Macrophages

Caco-2 cells (epithelial cells isolated from colon tissue derived from a 72-year-old, white male with colorectal adenocarcinoma) and RAW 264.7 cells (monocyte/macrophagelike cells originating from Abelson leukemia virus-transformed cell line derived from male BALB/c mice [40]) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Sigma-Aldrich Cat. No. 5671) supplemented with 10% heat-inactivated fetal bovine serum (Thermo Fisher Scientific Cat. No. 10–438-026), 1% Non-Essential Amino Acids (Sigma-Aldrich Cat. No. M7145), and 1% antibiotic/antimycotic solution (Thermo Fisher Scientific Cat. No. 15140122). All cells were maintained at 37 °C and 5% CO2. At the appropriate confluence, cells were passaged by washing with Dulbecco’s phosphate-buffered saline (DPBS) without calcium and magnesium followed by incubation with 0.25% Trypsin-EDTA at 37 °C for 3–5 min. Trypsin was neutralized via the addition of culture medium to the flasks and the cells were centrifuged at 100×g for 5 min to obtain a pellet. The cell pellet was resuspended in a fresh culture medium and titrated multiple times to obtain a cell suspension. The number of live cells was counted using the automated cell counter and the cells are thus seeded at the desired density.

Nanogel Cytotoxicity

The degree of cytotoxicity elicited by the nanoparticles was evaluated using the colorimetric CellTiter 96 AQueous One Solution Cell Proliferation Assay kit in which a soluble tetrazolium salt [3-[4,5-dimethylthiazol-2-yl]-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] (indicated as MTS) was bioreduced into a purple formazan product by viable cells; thus, the degree of bioreduction of the MTS reagent correlated to the number of healthy cells.

The nanoparticles were tested at different concentrations with Caco-2 and RAW 264.7 cells. The cells were plated at a density of 25,000 cells per well in 96-well plates 24 h prior to dosing with the nanoparticles. Lyophilized nanoparticles were UV irradiated for 30 min to kill any microbial agents prior to resuspension in cell culture medium at the required concentrations. After 24 h of exposure to nanoparticles, the cells were washed twice with DPBS and a solution of the assay medium premixed with the MTS reagent was added to each well. The plates were incubated for 2 h at 37 °C and 5% after which absorbance measurements were taken at 490 and 690 nm using a Biotek Cytation 3 Plate Reader.

Results and Discussion

Synthesis of Novel Cationic Nanoparticles with Dual Stimuli-Responsive Components

Nanocarriers based on 2-(diisopropylamino)ethyl methacrylate (DPAEMA) are promising candidates for siRNA transfection applications due to their lower cytotoxicity in comparison to nanogels formulated with the 2-diethylamino ethyl methacrylate (DEAEMA) as the primary cationic monomer. However, attempts at synthesizing nanoparticles based on DPAEMA as the sole cationic polymer proved to be challenging due to the lower pKa values of the polymer which causes a loss in the electrostatic charges required to maintain colloidal stability of the nanoparticles at neutral pH conditions (above pH 7.0) [41]. This work evaluates the parameters required to synthesize pH-responsive nanoparticles that exhibit both excellent cytocompatibility and robust colloidal stability at pH values above 7.0.

Co-polymers of DPAEMA and DEAEMA in combination with hydrophobic comonomers tert-butyl methacrylate (tBMA), cyclohexyl methacrylate (CHMA), and hexyl methacrylate (HMA) were synthesized via ARGET ATRP emulsion polymerization to generate pH-responsive cationic nanoparticles as shown in the schematic in Fig. 1. Each group of formulations were classified based on their non-charged hydrophobic comonomer and will be referenced henceforth as the CHMA, tBMA, and HMA series. Each series was composed of the following distribution of mol percentages of the monomers: 20% of the non-charged hydrophobic comonomer (i.e., CHMA, tBMA, or HMA), 20% of the polyethylene glycol surface graft, and 10–50% of either cationic comonomer to sum to a composition of 60% total cationic monomer (i.e. 50% DPAEMA + 10% DEAEMA and vice versa). The effects of increasing the incorporation of DPAEMA into the nanoparticle structures for each series is discussed.

Fig. 1.

Fig. 1

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Nanocarriers made from P(DEAEMA/DPAEMA-co-tBMA/HMA/CHMA-g-PEGMA) loaded with siRNA. The PEGMA graft induces a hydrophilic corona that allows for colloidal stability as well as biocompatibility; the DEAEMA/DPAEMA cationic monomers allow for pH responsiveness and siRNA complexation; and the tBMA/HMA/CHMA hydrophobic comonomers allow for tuning of the pH responsiveness and reduced cytotoxicity

Effect of Increasing DPAEMA Content on Nanoparticle Size, Volume Swelling Ratio, and Electrophoretic Potential

Purified nanogels were characterized using dynamic light scattering to determine the impact of increasing the DPAEMEA content. Each series included six compositions of DPAEMA which have been designated DP 0, DP 10, DP 20, DP 30, DP 40, and DP 50. DP 0 contains 60 mol% of DEAEMA and no DPAEMA, while DP 50 contains 50-mol% DPAEMA and 10-mol% DEAEMA. The collective results for these experiments are shown in “Comparisons Between the nanoparticles in Series Containing CHMA, tBMA, and HMA” section with comparisons of the various comonomers and in Figs. 2, 3, and 4. However, first, the results are shown for each individual co-monomer, where these individual figures are shown in our Supplemental Text, and the combined figures are shown as Figs. 2, 3, and 4.

Fig. 2.

Fig. 2

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Average hydrodynamic diameters (swollen and collapsed) of poly(DEAEMA-co-DPAEMA-co-CHMA/tBMA/HMA)-g-PEGMA nanoparticles synthesized via ARGET ATRP with increasing mol % composition of DPAEMA

Fig. 3.

Fig. 3

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Volume swelling ratios of poly(DEAEMA-co-DPAEMA-co-CHMA/tBMA/HMA)-g-PEGMA nanoparticles synthesized via ARGET ATRP with increasing mol % composition of DPAEMA. Each condition was measured in triplicate

Fig. 4.

Fig. 4

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Zeta potentials of poly(DEAEMA-co-DPAEMA-co-CHMA/tBMA/HMA)-g-PEGMA nanoparticles synthesized via ARGET ATRP with increasing mol% composition of DPAEMA

In general, the DPAEMA-based nanoparticles exhibited the desired pH-responsive behavior, maintaining a smaller hydrodynamic diameter at higher pH values which gradually increases as the pH is lowered (Figure S1). A shift in the onset of the swelling behavior toward lower pH conditions is also observed. These findings indicate that the lower pKa of the DPAEMA side group is playing a significant role in the charge interactions within the nanoparticle. This provides a unique avenue for tuning the responsiveness of the nanoparticles toward even more precise delivery of siRNA in unique pH microenvironments.

Copolymer Series Containing CHMA

Overall, hydrodynamic size measurements at pH 4.5 and pH 7.4 for the poly(DEAEMA-co-DPAEMA-co-CHMA)-g-PEGMA nanoparticles revealed an increase in nanoparticle diameter at both the swollen (pH 4.5) and collapsed (pH 7.4) configurations (Figure S2). These findings correlate with previous reports that monomers with bulkier side groups tend to form larger structures due to the steric hindrance present as these polymer chains are propagated during synthesis [42]. The collapsed hydrodynamic diameters of the poly(DEAEMA-co-CHMA)-g-PEGMA nanoparticles, without the addition of DPAEMA (DP 0) was 106.6 nm. This collapsed diameter did not show any significant changes until the addition of 30-mol% DPAEMA. Additional substitution with DPAEMA caused a 5% increase in the nanoparticle diameter for every 10-mol% increase in the composition of DPAEMA up to the largest collapsed diameter of 121.5 nm, which was obtained at 50 mol% DPAEMA. A similar trend was observed in the swollen diameters of these nanoparticle formulations; on average, a 5% increase in nanoparticle diameter was recorded for every 10-mol% increase in the DPAEMA content. Moreover, an increase in the swollen diameters are observed with DPAEMA compositions as low as the DP 10 formulations.

The impact of the more hydrophobic cationic monomer, DPAEMA, on the nanoparticles’ ability to imbibe water and ions in solution to facilitate swelling at low pH conditions was measured as a ratio of the volume of the nanoparticles in their swollen versus collapsed conformations, termed as the volume swelling ratio (VSR). Results indicate that increasing the amount of DPAEMA in the nanoparticles, while keeping the overall composition of cationic monomers constant, contributes to an increased volume swelling ratio (Figure S3). These results may be attributed to the increased steric hindrance introduced by the bulky side groups of DPAEMA, facilitating a larger pore structure within the nanoparticles and allowing agents to more easily diffuse.

The zeta potential values for the P(DEAEMA-co-DPAEMA-co-CHMA)-g-PEGMA nanoparticles were positive both at neutral and acidic pH conditions. The zeta potential at acidic pH ranged from 11.7 to 26.6 mV with the zeta potentials increasing as the composition of DPAEMA was increased (Figure S4). Overall, the zeta potential values at pH 7.4 remained below 5 mV but were all greater than 0 mV.

Copolymer Series Containing tBMA

DLS measurements of hydrodynamic size at pH 4.5 and pH 7.4 for the poly(DEAEMA-co-DPAEMA-co-tBMA)-g-PEGMA nanoparticles similarly showed a trend of increasing nanoparticle diameter across the swollen (pH 4.5) and collapsed (pH 7.4) configurations (Figure S5). For example, DP 0 formulations of the poly(DEAEMA-co-tBMA)-g-PEGMA nanoparticles exhibited a collapsed diameter of 114.78 nm and swollen diameter of 145.42 nm, while the DP 30 formulation presented a collapsed diameter of 106.60 nm and swollen diameter of 165.82 nm. The DP 0 formulations exhibit a 30.64-nm change in hydrodynamic diameter that is increased to a 59.22-nm difference with the addition of DPAEMA in the DP 30 formulations—a colossal 93% increase in the change in diameter of the nanoparticles. The aforementioned finding bolsters the conclusion that DPAEMA facilitates nanoparticle swelling; however, these benefits peak at the DP 30 formulations as a slight reduction in the volume swelling ratio is observed with higher DPAEMA formulations (i.e., DP 40 and DP 50) (Figure S6). The DP 40 and DP 50 nanoparticles, despite bearing a larger size, do not exhibit the same degree of volume swelling as the DP 30 formulations; the notable loss in swelling capacity may be attributed to the increased hydrophobic content of the particles that could hinder interactions with water and ions and dominate the favorable ionic and steric interactions that facilitated nanoparticle swelling.

The zeta potential values for the P(DEAEMA-co-DPAEMA-co-tBMA)-g-PEGMA nanoparticles were positive both at neutral and acidic pH conditions. The zeta potential at acidic pH ranged from 12.6 to 19.8 mV, increasing with the addition of DPAEMA until it peaked at the DP 30 formulation (Figure S7). At neutral conditions, the tBMA nanoparticles also exhibited zeta potential values less than 5 mV greater than 0 mV.

Copolymer Series Containing HMA

The hydrodynamic size measurements of the poly(DEAEMA-co-DPAEMA-co-HMA)-g-PEGMA nanoparticles at acidic and neutral pH conditions showed similar results to the previous two series of hydrophobic comonomers (i.e., CHMA and tBMA series). An increase in diameter of the nanoparticles was observed with higher nominal amounts of DPAEMA during synthesis (Figure S8). The average hydrodynamic diameter of the DP 0 formulations at acidic and neutral pH conditions were 137.18 nm and 108.83 nm, respectively. The average swollen hydrodynamic diameter of the nanoparticles increased by 41% to 193.63 nm with an increase the nominal amount of DPAEMA in the nanoparticles from 0 to 50 mol%. Similarly, the average collapsed hydrodynamic diameter of the HMA nanoparticles exhibited a 25% increase to reach a final diameter of 136.66 nm with the maximum amount of DPAEMA incorporations (i.e., DP 50).

The addition of the cationic monomer DPAEMA facilitates improved nanoparticle swelling evidenced by the rise in average volume swelling ratio from 2.02 to 3.79 with a 10 mol% incorporation of DPAEMA (Figure S9). However, these values drop slightly as higher ratios of DPAEMA are attained. For example, the VSR of the DP 50 HMA nanoparticles is only 3.00. These findings could be attributed to the effects of increased hydrophobicity in the nanoparticles with the presence of hexyl methacrylate, a six-carbon alkyl chain, in combination with the hydrophobic cationic monomer DPAEMA.

The zeta potential measurement for the HMA-based nanoparticles indicated the nanoparticles are cationic in nature with positive zeta potential values at both acidic and neutral pH conditions (Figure S10). Generally, in a neutral pH environment, the HMA nanoparticles exhibit zeta potential values approximately between 4 and 5 mV—charges slightly greater than the zeta potentials of the tBMA- and CHMA-based nanoparticles. These HMA nanoparticles also exhibited the highest zeta potential values of all formulations in the series with a maximum zeta potential value of 29.2 mV for the DP 30 formulation at pH 4.5.

Comparisons Between the Nanoparticles in Series Containing CHMA, tBMA, and HMA

Comparing the DLS results for the CHMA, tBMA, and HMA series of nanoparticles highlighted a unique trend in the nanoparticle hydrodynamic diameters across all series: the collapsed diameter of the nanoparticles was initially lowered by the addition of DPAEMA up to 20 mol% after which an increase in the nanoparticle diameter commenced up to the DP 50 formulations (Fig. 2). Consequently, these decreased diameters at the lower mol percentages of DPAEMA result in greater VSRs as these smaller nanoparticles are still capable of swelling to a similar or greater extent than the formulations without DPAEMA (Fig. 3).

The zeta potentials for all three-nanoparticle series at acidic pH (pH 4.5) were positive, confirming the cationic nature of the polymers. Overall, the HMA series of nanoparticles exhibited the highest electrophoretic potentials at neutral pH values but at acidic conditions, the CHMA series of nanoparticles exhibit the largest zeta potentials for DP 10, 20, and 30, respectively (Fig. 4). A positive charge imparts the nanoparticles with facile drug transfection capabilities and suggests the capability for strong cellular interactions with the negatively charged surface of tumors.

Spectroscopic Characterization of the Nanoparticles

Nanogels were purified, lyophilized, and degraded prior to analysis of monomer content. Overall, increasing the nominal feed concentration of DPAEMA led to a higher concentration of DPAEMA in the nanoparticle structure. The feed concentrations of DPAEMA were varied from 10 to 50 mol%. Figure 5 underscores the increase in the DPAEMA content as seen in the highlighted areas 1H NMR spectra. The DPAEMA peak at δ1.39 ppm becomes more prominent as we approach higher concentrations of DPAEMA; parallelly, decreased intensity of the DEAEMA peaks at δ1.30 and 3.19 ppm is observed. The PEGMA peak can also be observed at δ3.45 ppm confirming its successful incorporation into the nanoparticle structure.

Fig. 5.

Fig. 5

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Proton NMR spectra of P(DEAEMA-co-DPAEMA-co-CHMA)-g-PEGMA nanoparticles synthesized with increasing concentrations of DPAEMA in the feed. Samples were degraded with 10-mM TCEP in DMSO-d6 (δ 2.50) for 24 h. Spectra are normalized to the TMS peak at δ 0.00

Nanoparticle Cytocompatibility

Because of their small size, nanoparticles can easily enter the human body and even reach sensitive organs, even if those regions were not the original region of interest [43]. Studies have shown that after inhalation and through oral ingestion, NPs can be found widely distributed throughout the body, including the liver, heart, spleen, brain, lungs, and GI tract [44]. In addition, although different compositions will affect degradation rate, nanoparticles can remain in the body for a long time, with the estimated half-life of nanoparticles in human lungs to be 700 days [45]. Thus, because nanoparticles have such a systemic reach and have a prolonged time in vivo, toxicity is of the utmost concern when translating solutions into viable clinical treatments.

As cytotoxicity can be influenced by many different factors, including size, morphology, surface coating, and surface area [46], testing is paramount to ensure that the specific formulation of interest is safe, even if past formulations from literature have been shown to be non-cytotoxic, as toxicity data from tetrazolium based assays like MTT, MTS, and WST-1 can sometimes be inconsistent or even conflicting between studies [47]. Specifically, size is often the focus of nanoparticle delivery systems, because effective delivery is only achieved when the physical biological barriers are bypassed [48]. However, decreased nanoparticle size has been correlated with increased biological and chemical reactivity and thus increased cytotoxicity [49]. Thus, cytotoxicity studies are important also because of their role in shaping the design of the nanoparticle itself.

The biocompatibility of the novel DPAEMA containing nanoparticles was evaluated using an MTS assay. RAW 264.7 and Caco-2 cells were seeded in 96-well plates and exposed to the DP 10 to DP 50 CHMA series formulations of nanoparticles at varying concentrations. The cells were dosed for 24 h with the nanoparticles at the concentrations of 2.0, 0.8, 0.32, 0.128, 0.0512, and 0.0205 mg/mL.

The Caco-2 cells exhibited good cytocompatibility with the nanoparticles even at the highest concentration of 2 mg/mL (Fig. 6). Decreased cell viabilities (approximately 70%) were reported for the DP 10, 20, and 40 formulations and various concentrations. Conversely, some cell viabilities greater that 100% were observed in the DP 50 formulations which could indicate some stimulatory effect on the proliferation of cells. The RAW 264.7 cells showed greater susceptibility to nanoparticle toxicity with cell viabilities of 9.1% and 11.7% for the DP 10 and DP 20 formulations at 2 mg/mL (Fig. 7). Below 2 mg/mL, cell viabilities were generally above 70% except for one instance with the DP 30 formulation reporting a 57% cell viability at 0.8 mg/mL.

Fig. 6.

Fig. 6

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Percentage cell viability of Caco-2 cells after exposure to P(DEAEMA-co-DPAEMA-co-CHMA)-g-PEGMA nanoparticles at various concentrations for 24 h. % viability was measured using an MTS assay

Fig. 7.

Fig. 7

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Percentage cell viability of RAW 264.7 cells after exposure to P(DEAEMA-co-DPAEMA-co-CHMA)-g-PEGMA nanoparticles at various concentrations for 24 h. % viability was measured using an MTS assay

Conclusion

This work explores the use of DPAEMA, a cationic monomer, to synthesize improved siRNA transfection vectors for intracellular delivery. Nanoparticles composed of DEAEMA, DPAEMA, and non-cationic comonomers CHMA, tBMA, or HMA were evaluated for their unique physicochemical properties. Overall, higher amounts of DPAEMA in the nominal feed resulted in nanoparticles with larger diameters. Moreover, these nanoparticles elicited higher degrees of swelling relative to their non-DPAEMA counterparts which could facilitate greater nanoparticle loading efficiencies. Similarly, the electrophoretic potential values were elevated for higher incorporations of DPAEMA at acidic pH but did not show a significant difference between the compositions of DPAEMA at neutral values of pH. NMR spectra of lyophilized and degraded nanoparticles confirm the increasing amounts of DPAEMA incorporation into the nanoparticles with the most intense DPAEMA peak observed at δ1.39 ppm for the DP 50 formulations.

Nanoparticle formulations with the lowest amounts of DPAEMA exhibited significant cytotoxicity on Caco-2 and RAW 264.7 cells at the lowest concentrations tested (i.e., 2.0 and 0.8 mg/mL). The toxicity of these formulations can be attributed to the high amount of DEAEMA which has been observed to elicit cytotoxic effects. The addition of DPAEMA into the nanoparticles served to offset these toxicities as is evidenced by the higher cell viabilities in both Caco-2 and RAW 264.7 cells at even the highest concentrations, suggesting its capacity to enhance biocompatibility within the polymer Overall, these findings indicate that DPAEMA is an excellent candidate to serve as a comonomer for siRNA delivery vehicles.

Supplementary Material

supplement

Supplementary Information The online version contains supplementary material available at https://doi.org/10.1007/s44174-023-00122-8.

Acknowledgements

This research was supported in part by NIH Grant 1 R01-EB-000246-21. NAP’s work was also supported in part by the Office of the Dean of the Cockrell School of Engineering at The University of Texas at Austin for the Institute for Biomaterials, Drug Delivery, and Regenerative Medicine; and the UT-Portugal Collaborative Research Program. OLL’s work was supported by the University of Texas at Austin Provost Early Career Postdoctoral Fellowship Program.

Data Availability

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

supplement
NIHMS2204430-supplement-supplement.pdf (639.7KB, pdf)

Data Availability Statement

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

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