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. 2023 Sep 14;24(11):4958–4969. doi: 10.1021/acs.biomac.3c00630

Self-Immolative Polymer Nanoparticles with Precise and Controllable pH-Dependent Degradation

Samuel A Smith †, Bruna Rossi Herling §, Changhe Zhang †, Maximilian A Beach †, Serena L Y Teo §, Elizabeth R Gillies ‡, Angus P R Johnston §,*, Georgina K Such †,*
PMCID: PMC10649787  PMID: 37709729

Abstract

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Polymer nanoparticles have generated significant interest as delivery systems for therapeutic cargo. Self-immolative polymers (SIPs) are an interesting category of materials for delivery applications, as the characteristic property of end-to-end depolymerization allows for the disintegration of the delivery system, facilitating a more effective release of the cargo and clearance from the body after use. In this work, nanoparticles based on a pH-responsive polymer poly(ethylene glycol)-b-(2-diisopropyl)amino ethyl methacrylate) and a self-immolative polymer poly[N,N-(diisopropylamino)ethyl glyoxylamide-r-N,N-(dibutylamino)ethyl glyoxylamide] (P(DPAEGAm-r-DBAEGAm)) were developed. Four particles were synthesized based on P(DPAEGAm-r-DBAEGAm) polymers with varied diisopropylamino to dibutylamino ratios of 4:1, 2:1, 2:3, and 0:1, termed 4:1, 2:1, 2:3, and 0:1 PGAm particles. The pH of particle disassembly was tuned from pH 7.0 to pH 5.0 by adjusting the ratio of diisopropylamino to dibutylamino substituents on the pendant tertiary amine. The P(DPAEGAm-r-DBAEGAm) polymers were observed to depolymerize (60–80%) below the particle disassembly pH after ∼2 h, compared to <10% at pH 7.4 and maintained reasonable stability at pH 7.4 (20–50% depolymerization) after 1 week. While all particles exhibited the ability to load a peptide cargo, only the 4:1 PGAm particles had higher endosomal escape efficiency (∼4%) compared to the 2:3 or 0:1 PGAm particles (<1%). The 4:1 PGAm particle is a promising candidate for further optimization as an intracellular drug delivery system with rapid and precisely controlled degradation.

Introduction

Polymer drug delivery systems have generated significant interest for a variety of biomedical applications. They have demonstrated the capability to aid the delivery of poorly soluble drugs, protect sensitive cargo, enhance target specificity, and reduce side effects.1 In particular, nanoparticle delivery systems are crucial to the delivery of emerging biopharmaceutical therapeutics, such as those based on mRNA, DNA, and proteins, due to their protection from degradation and enhanced intracellular delivery.2 However, the number of clinically approved nanoparticle delivery systems remains low, and the poor efficiency of delivery at crucial steps, such as endosomal escape, remains an area for improvement.3,4 In addition, the development and understanding of novel nanoparticles is crucial to enable the delivery system to be tailored for particular applications.5,6

An effective strategy to facilitate controlled release from the delivery system at a specific location is through the use of stimuli responsive materials, which respond to the environmental changes present at the target site.7 In particular, pH is an important endogenous biological stimulus as acidic pH is associated with the tumor microenvironment (pH 6.5–7.0)8,9 and the maturation of the endocytosis pathway (pH 6.8 to <5.0), which are of particular interest for vaccine development and anticancer therapies.10,11 However, the absence of polymer degradation at the end of life is a limitation for numerous in vivo applications including vaccine development, diagnostics, and drug delivery.12 For example, the commonly used polymeric transfection agent polyethylenimine (PEI) has been shown to induce toxicity through off target destabilization of the plasma and nuclear membrane.13 −15 Additionally, the cationic lipid component of the current mRNA nanoparticle vaccines has been observed to have a ∼40-day half-life and was found to induce low levels of immune activation over that time period.6 Therefore, precisely controlling polymer degradation after the nanoparticle has performed its function is an important parameter to consider in the design of polymer nanoparticles for biomedical applications.

One strategy to precisely control the degradation of the polymer delivery system is through the use of self-immolative polymers (SIPs). SIPs are a class of polymers that undergo end-to-end depolymerization in response to stimuli.16 Above their ceiling temperature, SIPs exist in a metastable polymer form due to the presence of an end-cap that provides a barrier to depolymerization. Upon removal of the end-cap, depolymerization to the monomer is favored, and thus end-to-end depolymerization occurs. A range of SIPs have been developed that have been shown to respond to a variety of endogenous and exogenous stimuli, including pH, redox, temperature,17 and metals,18 as well as light and enzymes.19−22

Recently, a number of SIP nanoparticles with applications for drug delivery have been developed. Gillies and co-workers developed self-immolative poly(ethyl glyoxylate)s (PEtGs) that depolymerize into glyoxylic acid, an intermediate that can be processed in the liver.23 Nanoparticles composed of the triblock copolymer poly(ethylene glycol) (PEG)-b-PEtG-b-PEG were constructed with linker end-caps between the blocks that were responsive to UV light, reducing agents, and hydrogen peroxide. For example, PEG-b-PEtG-b-PEG particles were found to disassemble through the depolymerization of the PEtG block in ∼7 h in response to 10 mM dithiothreitol (DTT), thereby enhancing the release of model drugs. In another study, Wender, Waymouth, and co-workers developed polymer particles of (oligo(carbonate-b-α-amino ester) as a charge-altering releasable transporter (CART) system.24,25 The depolymerization of the oligo(α-amino ester) effectively released encapsulated mRNA at pH 7.4, and degraded into a nontoxic degradation product (dimerized serine diketopiperazine). However, the cyclization reaction during the depolymerization of the CART polymer is inhibited by protonation of the pendent amine at low pH, so while the depolymerization and release was very effective at pH 7.4, it was very minimal at pH 6.8–5.0.

Self-immolative nanoparticles that depolymerize under low pH conditions have been developed using poly(glyoxylamide) (PGAm).26 For example, nanoparticles composed of the block copolymer PEG-b-poly(N,N-(diisopropylamino)ethyl glyoxylamide) (PEG-b-DPA-PGAm) with an acid-responsive 4-methoxytrityl (MMT) end-cap linker underwent 20% depolymerization at pH 7.4 and 40% depolymerization at pH 5.0 after 24 h. In this system, the pH-selective depolymerization was achieved by tuning the reactivity of the end-cap as the end-cap was positioned at the interface between the PEG and DPA-PGAm blocks and its cleavage was influenced by the hydrophobicity of the PGAm block, which depended on pH. However, the nanoparticles were not highly stable at pH 7.4. A similar response to pH was observed with DPA-PGAm polymers with a star architecture and the MMT end-cap, when included in the particle core of the core–shell particles stabilized by PEG-b-poly(N,N-(diethylamino)ethyl methacrylate-r-N,N-(diisopropylamino)ethyl methacrylate) (PEG-b-P(DEAEMA-r-DPAEMA)) as the shell polymer.27 The high percentage of DPA-PGAm depolymerization at pH 7.4 was attributed to the migration of the DPA-PGAm to the particle surface as the amount of protonation of the pendent tertiary amines increased. As a result, it was postulated that increasing the hydrophobicity of the amine pendants of the core polymer materials could potentially decrease the proton access to the core of the nanoparticles.

The work described here aimed to precisely design PGAm polymers with optimized pH response for biological delivery applications. To design a system that effectively controlled the onset of depolymerization, the reactivity of the end-cap, the access of the end-cap to the cleaving stimulus, and the rate of end-to-end depolymerization post end-cap removal were all parameters that were considered in the material design. For SIP backbones such as polycarbamates, that depolymerize by cyclization reactions, the rate of cyclization is the rate-determining step of depolymerization.28 In contrast, the rapid depolymerization mechanisms of polyacetals and poly(benzyl ethers) mean the end-cap cleavage is the rate-determining step.29 End-cap cleavage is influenced by both the reactivity of the end-cap and the access of the stimuli to the end-cap.19,30 While extensive research has focused on tuning the onset and rate of depolymerization through the end-cap reactivity, the differences in onset, rate, and amount of depolymerization between two pH environments has so far been marginal within the narrow range of pH 7.4–5.0 (present in biological systems).19 Therefore, there remains a need for generating a tunable depolymerization transition with pH through the combined control of the end-cap reactivity with the end-cap access. In the previously mentioned studies, DPA-PGAm nanoparticles were found to be unstable and undergo polymer migration at pH 7.4. Herein, a series of poly[N,N-(diisopropylamino)ethyl glyoxylamide-r-N,N-(dibutylamino)ethyl glyoxylamide] P(DPAEGAm-r-DBAEGAm) (PGAm) polymers with a 4:1, 2:1, 2:3, and 0:1 ratio of diisopropylamino:dibutylamino (DPA:DBA) were synthesized. The PGAms were combined with the pH-responsive and amphiphilic polymer PEG-b-PDEAEMA to form nanoparticles with diameters of 150–200 nm. The pH of particle disassembly was carefully tuned from pH 7.4 to 5.0 by adjusting the ratio of the DPA and DBA pendent groups. Importantly, the PGAm particles showed minimal depolymerization of less than 10% at pH 7.4 after ∼ 2 h and 20–50% depolymerization at pH 7.4 after 1 week, but extensively depolymerized (60–80%) within 2 h below the pH of particle disassembly. The ability of these delivery systems to load and release a peptide cargo was investigated by using a small peptide–polymer conjugate (HiBiT), which can also be used to assess endosomal escape quantitatively. The 4:1 PGAm, 2:1 PGAm, and 2:3 PGAm particles demonstrated effective loading and release of a HiBiT peptide polymer conjugate. The endosomal escape of these self-immolative systems was investigated by the Split Luciferase Endosomal Escape Quantification (SLEEQ) assay.31,32 Endosomal escape is an important bottleneck for therapeutic delivery especially for biological cargo and thus there is a need to optimize this behavior in new delivery systems. Higher levels of endosomal escape were observed for the 4:1 PGAm system with ∼4% of peptide delivered to the cell transported to the cytosol, compared to <1% delivered to the cytosol for the other systems. This aligned with our previous studies on the optimal pH of particle disassembly for endosomal escape.13 Overall, the nanoparticles described here are simple and modular and offer significant potential for the controlled release of biological cargo.

Materials and Methods

Materials

1-Bromobutane (98+%), 1,4-dioxane (ACS reagent, ≥99.0%), ethyl glyoxylate (50% in toluene), ethylenediamine (99%), and phosphorus(V) pentoxide (98%) were purchased from Alfa Aesar and used as received. 4,4′-Dimethoxytrityl chloride (DMT) (95%), potassium chloride (BioReagent, ≥99%), potassium phosphate monobasic (BioReagent, ≥99%), sodium chloride (BioReagent, ≥99%), sodium phosphate dibasic (BioReagent, ≥99%), tetrahydrofuran-d8 (≥99.5 atom % D), trifluoroacetic acid (ReagentPlus, 99%), and maleic acid were purchased from Sigma-Aldrich and used as received. Di-tert-butyl dicarbonate (99%) and N,N-diisopropylethylenediamine (97%) were purchased from Oakwood Chemicals and used as received. Dichloromethane (RCI Premium), dimethylformamide (RCI Premium), hexane (95%, RCI Premium), magnesium sulfate (AR), methanol (AR), potassium carbonate (AR), sodium bicarbonate (AR), sodium hydroxide (AR), and tetrahydrofuran ((stabilized with BHT, RCI Premium) were purchased from Chem Supply and used as received. Ethyl acetate (AR) and triethylamine (TEA) (AR) were purchased from Ajax Fine Chemical, and 1,4-dithiothreitol was purchased from Astral Scientific and used as received. N,N-(Diethylamino)ethyl methacrylate (99%) and N,N-(diisopropylamino)ethyl methacrylate (97%) were purchased from Sigma-Aldrich and passed through basic alumina prior to use. Anhydrous TEA and dichloromethane were obtained through distillation over CaH2 under nitrogen gas before use, while anhydrous N,N-dimethylformamide (DMF) was obtained through storage with activated molecular sieves (4 Å, Ajax Fine Chemical).

Lacey Formvar/carbon-coated 300 mesh copper grids were purchased from ProSciTech, 3.5 kDa MWCO dialysis “snakeskin” bag was purchased from Thermo Fisher Scientific, Float-A-Lyzer tubes (100 kDa MWCO, 5 mL) were purchased from Sigma-Aldrich, and Millex syringe filters with poly(ether sulfone) (PES) membrane (0.45 μm pore diameter) were purchased from Merk Millipore and used according to the manufacturer’s instructions. The SLEEQ assay components were sourced from the Nano-Glo live cell assay system except for the HiBiT peptide (CGGSGGSGGVSGWRLFKKIS) that was purchased from GL BioChem and LgBiT protein that was expressed and purified from Escherichia coli using the pBiT1.1-N[TK/LgBiT] vector (Promega). AlamarBlue reagent was purchased from Thermo-Fisher Scientific, and Dulbecco’s modified Eagles medium (DMEM; Thermo-Fisher Scientific) was diluted 20% in Milli-Q water to make up the media. To obtain 10 mM phosphate buffered saline (PBS), 0.1 M PBS stock solution was prepared using sodium chloride (80 g, 137 mmol), potassium chloride (2 g, 27 mmol), potassium phosphate monobasic (2.4 g, 17 mmol), and sodium phosphate dibasic (14.4 g, 10 mmol) in reverse osmosis water (1.0 L) and the 0.1 M PBS stock was diluted by a factor of 10 to be 10 mM before use. PEG-b-PDEAEMA and poly[N,N-(diethylamino)ethyl methacrylate-r-N,N-(diisopropylamino)ethyl methacrylate-random-(pyridyl disulfide)ethyl methacrylate] P(DEAEMA-r-DPAEMA-r-PDSEMA) were synthesized following previously reported protocols.25

Characterization

1H and 13C NMR spectroscopy was performed on a Varian 400 MHz (100 MHz for 13C) NMR Spectrometer (Varian, Inc., United States of America) operated at ambient temperature. Gel permeation chromatography (GPC) was run on a Shimadzu modular system fit with Waters HT3 and HT4 columns (10 μm bead size), a Wyatt Dawn DSP multiangle light scattering detector (690 nm, 30 mV), a Wyatt OPTILAB EOS interferometric refractometer (690 nm), and a UV–vis detector (310 nm). The mobile phase was tetrahydrofuran (HPLC grade) set to a flow rate of 0.3 mL/min. The molecular weight was determined by comparison to poly(methyl methacrylate) (PMMA) standards. Nanoparticles were analyzed by dynamic light scattering (DLS) on a Horiba nanopartica SZ-100 series nanoparticle analyzer (Horiba Scientific, Japan) with the scattered light set to be detected at 90° and on a NanoSight NS300 (Malvern Instruments ltd., United Kingdom; NTA) fitted with a 638 nm laser and a sCMOS camera. The nanoparticle tracking analysis (NTA) was run using a camera level set 11 and a detection threshold set to 5. CryoTEM images were taken using a Talos L120C Transmission Electron Microscope (FEI Company, Inc., United States of America) operating at an acceleration voltage of 120 keV, located at the Melbourne Advanced Microscope Facility (Bio21 Molecular Science and Biotechnology Institute, the University of Melbourne). Imaging was performed using low dose mode (∼1000 electrons per nm2) using 22000× magnification and a defocus of ∼−13 μm. Luminescence spectroscopy was performed on the IVIS Lumina II (PerkinElmer) with an exposure time of 10 s. Fluorescence spectroscopy was performed on a Cary Eclipse fluorescence spectrophotometer (Agilent Technologies, Inc.) with an excitation set to 630 nm and the emission recorded at 661 nm, with an excitation and emission slit width set to 10 nm.

Synthesis

Synthesis of poly[N,N-(diisopropylamino)ethyl glyoxylamide-r-N,N-(dibutylamino)ethyl glyoxylamide] P(DPAEGAm-r-DBAEGAm) polymers.

General Synthesis of PEtG-DMT

PEtG end-capped with 4,4′-dimethoxytrityl (DMT) was synthesized following a previously reported protocol.15,231H NMR (400 MHz, CD3CN): δ 7.53–6.8 (m, Ar-H), 5.75–5.48 (m, −OCH(CO2Et)O−), 4.23 (brs, −C(O)OCH2CH3), 1.30 (brs, −CH2CH3). Mn = 42.5 kDa (NMR), 42.0 kDa (GPC). Đ = 1.34 (GPC).

General Synthesis of P(DPAEGAm-r-DBAEGAm)

A representative synthesis of P(DPAEGAm-r-DBAEGAm) (2:3) is added here in the main text and was based on a previously reported protocol.20 The other polymers in the series are detailed in the Supporting Information. PEtG-DMT (150 mg, 1.5 mmol of repeating units) was dissolved in anhydrous 1,4-dioxane (1.5 mL) in a flame-dried Schlenk flask. To this solution, N,N-diisopropylethylenediamine (158 mg, 1.1 mmol) and N,N-dibutylethylenediamine (569 mg, 3.3 mmol) were added at the same time and the reaction was stirred for 5 days under nitrogen at ambient temperature. Afterward, the solvent was removed under reduced pressure, and the polymer was purified by precipitation into acetonitrile. Upon drying, P(DPAEGAm-r-DBAEGAm) was obtained. 1H NMR was used to characterize the modification efficiency and pendant group ratio, setting the integration of acetyl protons at the backbones as 1. 1H NMR (400 MHz, CDCl3): δ 8.98–7.60 (brs, NH, 1H), δ 5.60–5.80 (brs, −OCHO–, 1H), δ 3.20 (brs, −NHCH2–2H, 2H), δ 2.98 (brs, −CH(CH3)2, 1H), δ 2.55 (brs, −NHCH2CH2NR2–, 2.4H), δ 2.43 (s, NCH2CH2CH2CH3, 2.4H), δ 1.39 (s, butyl −NHCH2CH2CH2CH3, 2.4H), δ 1.27 (brs, butyl −NHCH2CH2CH2CH3, 2.5H), δ 1.00 (brs, isopropyl −CH3, 4.8H), δ 0.89 (t, butyl −CH3, 3.6H). Mn = 90.2 kDa (1H NMR).

Synthesis and Characterization of PGAm Nanoparticles

Synthesis of PGAm Nanoparticles

The polymers PEG-b-PDEAEMA and P(DPAEGAm-r-DBAEGAm) were dissolved in THF to form separate stock solutions (10 mg/mL). PEG-b-PDEAEMA (100 μL, 10 mg/mL) and P(DPAEGAm-r-DBAEGAm) (200 μL, 10 mg/mL) solutions were mixed and then precipitated as a single addition (1 × 300 μL) into PBS buffer solution at pH 8.0 (3.0 mL) that was stirred at 1000 rpm at 25 °C. The particle suspension was then stirred at 300 rpm for 15 min at 25 °C and then dialyzed in a 100 kDa MWCO dialysis tube against PBS pH 8.0 (24 h, 6 buffer changes, 25 °C). The nanoparticles were then removed, stored at 25 °C for approximately 36 h, and filtered through a 0.45 μm PES filter before use.

Characterization of PGAm Nanoparticles by Dynamic Light Scattering (DLS)

A consistent volume of particles was used for all of the DLS analyses. Assuming no loss during filtration, one could calculate from the given procedures that the concentration was ∼0.15 mg/mL. The PGAm nanoparticles (150 μL) were added to filtered and preheated PBS buffer from pH 8.0 to 6.0 (1.0 mL) and incubated for 2 min at 37 °C before measurement by DLS at 37 °C. For the stability studies at pH 7.4, the PGAm nanoparticles (150 μL) were added to filtered and preheated PBS buffer pH 7.4 (1.0 mL) and was incubated for 1 to 196 h at 37 °C. Data from the intensity distributions are reported.

Depolymerization Assay of PGAm Nanoparticles by 1H NMR Spectroscopy

The deuterated PBS (dPBS) buffer was used for 1H NMR tracking of the depolymerization in nanoparticles. NaCl (96.0 mg, 1.7 mmol), Na2HPO4 (17.3 mg, 0.12 mmol), KH2PO4 (2.88 mg, 0.02 mmol), KCl (2.40 mg, 0.03 mmol), and maleic acid (5.26–5.37 mg, 45–46 μmol) were dissolved in deuterium oxide (12.0 mL) and adjusted to pH 8.0 using 0.1 and 1.0 M deuterated NaOH (0.18–0.66 mL) to make 1× deuterated PBS pH 8.0 (dPBS). The polymers PEG-b-PDEAEMA and P(DPAEGAm-r-DBAEGAm) were dissolved in deuterated tetrahydrofuran (THF-d8) to form separate stock solutions (20 mg/mL). PEG-b-PDEAEMA (100 μL, 20 mg/mL) and P(DPAEGAm-r-DBAEGAm) (200 μL, 20 mg/mL) solutions were mixed and precipitated as a single addition (1 × 300 μL) into dPBS pH 8.0 (3.0 mL) that was stirred at 1000 rpm at 25 °C. The particle mixture was then stirred at 300 rpm for 15 min at 25 °C then placed under reduced pressure for 5 min. The nanoparticles were stored at 25 °C for ∼24 h, and then the pH was adjusted using 0.1 M deuterated HCl solution using an InLab Semi-Micro pH sensor (Mettler Toledo) at 37 °C. The particles were measured over time by 1H NMR spectroscopy and stored at 37 °C between measurements.

Synthesis of HiBiT Conjugate

The HiBiT Conjugate was synthesized following a previously reported protocol.25 The conjugate was purified via dialysis in a 10 kDa MWCO bag against PBS pH 6.0 (1 L, 5 buffer changes) for 2 days at ambient temperature and 2 days at 4 °C.

Loading the HiBiT Conjugate into (PDPAEGAm-r-DBAEGAm) Nanoparticles

The polymers PEG-b-PDEAEMA and P(DPAEGAm-r-DBAEGAm) were dissolved in THF to form separate stock solutions (10 mg/mL). The HiBiT conjugate (100 μL) was added to a stirred solution of PBS pH 8.0 (2.9 mL), and immediately, the mixed solution of PEG-b-PDEAEMA (100 μL, 10 mg/mL) and P(DPAEGAm-r-DBAEGAm) (200 μL, 10 mg/mL) solutions was precipitated as a single addition (1 × 300 μL) and stirred at 1000 rpm at 25 °C. The particle mixture was then stirred at 300 rpm for 15 min at 25 °C and then dialyzed in a 100 kDa MWCO dialysis tube (5 mL) against PBS pH 8.0 (24 h, 6 buffer changes, 25 °C). The nanoparticles were then removed, stored at 25 °C for ∼36 h, and then filtered through a 0.45 μM PES filter.

Release of the HiBiT Conjugate from PGAm Particles

The elution fractions E3 and E4 of the Sepharose 6B column-purified HiBiT-conjugate-loaded PGAm nanoparticles were combined and diluted by a factor of 10 in PBS at pH 8.0. The preparation of the column is highlighted in the Supporting Information. The diluted nanoparticles (25 μL), purified largeBiT (LgBiT, 50 μL, 50 nM in PBS pH 7.4–5.0), and Nano-Glo live cell substrate (25 μL, 1/20 in the dilution buffer according to the manufacturer’s instructions) were combined, and the luminescence was measured after 12 min using the IVIS Lumina II.

Cell Viability

The alamarBlue assay was used to test the cell viability and was performed on the combined fractions E3 and E4 of the Sepharose 6B column-purified HiBiT-Cy5-conjugate-loaded PGAm particles following a previously reported protocol at 0.12, 0.23, 0.5, 0.9, 1.9, 3.8, 7.5, 15, and 30 × 109 particles/mL.25

SLEEQ Assay

The SLEEQ assay was performed on the HiBiT-Cy5-conjugate-loaded PGAm particles following a previously reported protocol.25 The combined fractions E3 and E4 of the Sepharose 6B column-purified HiBiT-Cy5-conjugate-loaded PGAm particles were diluted to 2.5 × 109 particles/mL in media and added to HEK293 cells stably expressing LgBiT-SNAP-actin (LSA) constructs for 4 h. After subtraction of the supernatant signal from the live signal, all repeats of the 0:1 PGAm particles and one repeat of the 2:3 PGAm particle were found to produce a negative value. This was due to the low, baseline level readout of cytosolic luminescent signal for the particles. As a negative cytosolic luminescent value was not possible, the cytosolic luminescent signal and the escape efficiency of three repeats of the 0:1 PGAm particle and one repeat of the 2:3 PGAm particle was set to zero.

Results and Discussion

The initial PEtG precursor polymer was synthesized through the anionic polymerization of ethyl glyoxylate in anhydrous dichloromethane at −20 °C and end-capped with an acid labile 4,4′-dimethoxytrityl (DMT) end-cap.15 The DMT end-cap was selected as previous work demonstrated that DMT end-capped polymers had the fastest depolymerization rate in an acidic pH, and therefore, the breakdown of the delivery system and cargo release was likely to be rapid.15 The PEtG-DMT polymer was found to be 42.5 and 42.0 kDa by 1H NMR spectroscopy and GPC, respectively, with a moderate and unimodal dispersity (Đ) of 1.34 (Figure S1).

The P(DPAEGAm-r-DBAEGAm) polymers were synthesized through postpolymerization modification of PEtG with varied ratios of N,N-dibutylethylenediamine (DBA) and N,N-diisopropylethylenediamine (DPA). To synthesize DBA, the ethylenediamine precursor was first monoprotected with Boc, then alkylated with 1-bromobutane on the amine group in DMF, and finally deprotected with TFA (Figures S2 and S3). The DBA product was confirmed through 1H and 13C NMR spectroscopy (Figure S4). The tert-butyl N,N-dibutylaminoethylcarbamate intermediate was purified through column chromatography to separate the product from the monoalkylated side product and the tert-butanol impurity present in the tert-butyl 2-aminoethylcarbamate precursor. To attach the diamines to the side chain of the polymer, PEtG was dissolved in anhydrous 1,4-dioxane and reacted with excess diamine at varied ratios of DPA to DBA.20 A library of four P(DPAEGAm-r-DBAEGAm) polymers were synthesized. The DPA:DBA ratios of the polymers were determined by 1H NMR spectroscopy, through the comparison of the integration of the terminal methyl groups of the isopropyl and the butyl moieties (Figures S5–S8). The DPA:DBA ratios of the four P(DPAEGAm-r-DBAEGAm) were found to be 4:1, 2:1, 2:3, and 0:1. The molar masses of the polymers were based on the number of repeating units of and the average molecular weight of repeating unit for each P(DPAEGAm-r-DBAEGAm) polymer, as the P(DPAEGAm-r-DBAEGAm) polymers were found to adhere to the GPC column.23 The Mn for the 4:1, 2:1, 2:3, and 0:1 P(DPAEGAm-r-DBAEGAm) polymers were calculated to be 85.2, 87.2, 90.2, and 94.7 kDa, respectively.

The P(DPAEGAm-r-DBAEGAm) polymers were combined with a pH-responsive PEG-b-PDEAEMA amphiphilic shell polymer to form pH responsive nanoparticles (Figure 1).33,34 These particles will be termed 4:1 PGAm, 2:3 PGAm, 2:1 PGAm, and 0:1 PGAm particles. They were formulated via nanoprecipitation from THF solutions into PBS pH 8.0 and characterized by DLS, NTA and cryoTEM (Figures 2, S13, S14, and S22). The side chain composition of the PGAm polymer was found to change the size and distribution of the PGAm particles (Table 1). The 4:1 PGAm particles were found to have a mean diameters of ∼220 and ∼160 nm by DLS and NTA, respectively. The 4:1 PGAm particles had a PDI < 0.2 reported by DLS. Similarly, the 2:1 PGAm particles were found to have a mean diameter of ∼230 and 160 nm by DLS and NTA respectively with a PDI < 0.2. The mean diameter of the 2:3 PGAm particles were found to be ∼240 and ∼180 nm by DLS and NTA, respectively. The size distribution of the 2:3 PGAm particles was found to be disperse and resolved into 3 general populations around 110, 150, and 220 nm in NTA. However, the PDI was still less than 0.2 when measured by DLS. In contrast, the distribution of the 0:1 PGAm particles was found to have a symmetrical and monomodal distribution, with a mean diameter of 150 ± 60 nm (2σ) by NTA, and PDI < 0.2 reported by DLS. In summary, the library of the four PGAm particles were synthesized with a mean diameters ranging from 150–240 nm.

Figure 1.

Figure 1

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(A) General schematic of the synthesis of the PGAm polymers from ethyl glyoxylate (EtG). Four polymers with varying ratios of DPA to DBA (4:1, 2:1, 2:3, and 0:1 respectively) were synthesized (Figures S5–S8). (B) Schematic of the PGAm particle formulation. General schematic of the disassembly and depolymerization of the PGAm particles composed of the SIP poly[N,N-(diisopropylamino)ethyl glyoxylamide-r-N,N-(dibutylamino)ethyl glyoxylamide] (PDPAEGAm-r-PDBAEGAm) polymer with the acid-responsive end-cap DMT and the pH-responsive stabilizer poly(ethylene glycol)-b-poly(N,N-(diethylamino)ethyl methacrylate) (PEG-b-PDEAEMA) at a 2:1 (w/w) ratio.

Figure 2.

Figure 2

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PGAm nanoparticles show pH tunable disassembly. Plot of the frequency against particle diameter (nm) from the DLS intensity distributions (triplicate samples) of the (A) 4:1 PGAm particle, (C) 2:1 PGAm particle, (E) 2:3 PGAm particle, and (G) 0:1 PGAm particle in PBS pH 8.0 measured by DLS at 37 °C. Representative CryoTEM images of the (B) 4:1 PGAm particle, (D) 2:1 PGAm particle, (F) 2:3 PGAm particle, and (H) 0:1 PGAm particle in PBS pH 8.0. Plot of (I), the mean particle diameter (nm) against pH and (J) the mean particle diameter (nm) over time (hr) in PBS pH 7.4 at 37 °C for the 4:1 (red, downward triangle), 2:1 (blue, upward triangle), 2:3 (black, circle), and 0:1 (yellow, square).

Table 1. Characterization of the PGAm Nanoparticles.

particles size(DLS)/nm PDI(DLS) size(NTA)/nm morphology/cryoTEM
4:1 220 ± 20 <0.2 160 spherical
2:3 240 ± 5 <0.2 180a mixedb
2:1 230 ± 4 <0.2 160 mixedc
0:1 190 ± 2 <0.2 140 spherical
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a

Size resolved to be a mixture of 110, 150, and 220 nm in NTA.

b

Predominately spherical particles with some irregular shaped particles.

c

A mixture of the irregular particles and well-defined spherical particles.

Interestingly, imaging by cryoTEM revealed that the side chain composition of the PGAm polymer altered the morphology of the PGAm particles (Figures 2 and S13), with nonspherical particles being observed for the 2:1 and 2:3 particles. The analysis by cryoTEM suggested that the mean size information collected by DLS and NTA for the 2:1 and 2:3 PGAm particles was problematic due to the presence of aspherical particles. Thus, the mean size reported for the 2:1 and 2:3 PGAm particles was used as an indicator to monitor change, rather than an accurate measure of particle diameter.

To understand the particle behavior in response to the pH decrease that would occur during endocytosis, the nanoparticles were incubated in PBS solutions adjusted to pH 8.0–5.0 and were measured by DLS (Figure S20). In the case of the 4:1 system, the nanoparticles maintained a mean diameter of ∼220 nm and a consistent PDI and count rate from pH 8.0 to 7.2 (Figures 2I and S9). However, at ≤pH 7.0, the count rate value dropped to baseline levels and the DLS reported a compromised autocorrelation function that indicated that the 4:1 nanoparticle had disassembled into soluble unimers.34 Therefore, the disassembly pH of the 4:1 nanoparticle was found to be pH 7.0. As the ratio of DBA to DPA increased, the disassembly pH of the particles was found to decrease. The 2:1 PGAm particles had a disassembly point of pH 6.4, while the 2:3 and 0:1 particles had a disassembly point of pH 6.0 and <5.0, respectively (Figures 2I and S10–S12). This trend is due to the transition of the polymer backbone from hydrophobic to hydrophilic based on the protonation of the amine. This transition is lower for the polymer with more DBA due to the greater steric hindrance and hydrophobicity of the dibutyl groups compared to the diisopropyl groups. We have observed similar trends in our earlier work on these polymers.35

To understand the stability of the PGAm particles at physiological pH, the particles were incubated in PBS pH 7.4 and analyzed by DLS over time (Figure 2J). The 2:1, 2:3, and 0:1 PGAm particles were observed to maintain the mean particle diameters, PDI and count rates, indicating high particle stability over a period of 1 week at 37 °C. In contrast the 4:1 PGAm particle was observed to maintain a similar PDI and count rate, but slightly increased in mean particle size after 24 h and then remained a similar size for a period of 1 week.

To understand the depolymerization of the P(DPAEGAm-r-DBAEGAm) polymers in response to a pH decrease, the PGAm nanoparticles were formed in deuterated PBS, adjusted to pH 7.4 and a pH below the particle disassembly pH for the particular system, then depolymerization was measured quantitatively by 1H NMR spectroscopy (Figures S15–S18). In acidic environments poly(glyoxylamide) and poly(ethyl glyoxylate) polymers with a DMT end-cap have been observed to undergo depolymerization (Figure 3A).14,23,35 The P(DPAEGAm-r-DBAEGAm) polymer is expected to exhibit similar depolymerization behavior to other PGAm and PEtG polymers with the same DMT end-cap. If depolymerization had occurred, then the acetal proton of the degradation product glyoxylamide hydrate 2,2′-dihydroxyacetamide (highlighted in Figure 3) would be observed as a singlet at δ 5.3 ppm in the 1H NMR spectra. In contrast, if the polymer was not degraded but solvated, then a broad peak from δ 5.5–5.8 ppm would be observed. If the polymer remained in the particle, then no peak would be observed due to the short T2 or spin–spin relaxation time. To quantify the extent of depolymerization, the integration of the singlet at δ 5.3 ppm of the glyoxylamide hydrate was compared to a maleic acid internal standard. This experiment was repeated twice (Figures 3, S19, and S28).

Figure 3.

Figure 3

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PGAm nanoparticles show controlled depolymerization behavior with pH. (A) Mechanism of the end-cap cleavage and depolymerization of the P(DPAEGAm-r-DBAEGAm) polymers. Plot of the depolymerization over time for the (B) 4:1 PGAm particle at pH 7.4 (blue, circle) and pH 6.8 (red, triangle), (C) 2:1 PGAm particle at pH 7.4 (blue, circle) and pH 6.0 (red, triangle), (D) 2:3 PGAm particle at 7.4 (blue, circle) and pH 5.8 (red, triangle), and (E) 0:1 PGAm particle at pH 7.4 (blue, circle) and pH 5.0 (red, triangle), measured by 1H NMR. The samples were incubated at 37 °C between measurements by 1H NMR at ambient temperature. Experiment was performed with n = 2 (Figure S28).

Minimal depolymerization of the P(DPAEGAm-r-DBAEGAm) (2:1) polymer was observed at pH 7.4, with ∼1% of the hydrate degradation product observed after ∼2 h and ∼20% observed after 1 week (Figure 3C). In contrast, extensive depolymerization was observed at pH 6.0, with ∼78% of the degradation product observed after 2 h, and then it remained unchanged for a period of 1 week. Similarly, the 4:1, 2:3, and 0:1 PGAm particles underwent minimal depolymerization at pH 7.4 after ∼ 2 h with ∼5%, 1%, and 6% depolymerization observed, respectively. This stability was maintained for at least 1 week at pH 7.4 with depolymerization of the 4:1, 2:3, and 0:1 PGAm particles being ∼18%, 19%, and 46% respectively (Figure 3B, D, and E). In contrast, 72–79% depolymerization was observed after 2 h below the disassembly pH for each of the 4:1, 2:3, and 0:1 PGAm particles, and again, the depolymerization percentage was maintained for a period of 1 week. Interestingly, the complete generation of the hydrate degradation product in solution was not observed. This could be due to some nanoparticles remaining in the system, possibly stabilized by hydrophobic components from the depolymerized building blocks, or material stuck to the surface of the pH sensor used to adjust pH. Overall, the results of the depolymerization assay indicated high stability of the nanoparticle at pH 7.4. In contrast, upon the particle internalization into acidic endosomal compartments, extensive depolymerization would be expected to occur within 1–2 h. These results are an initial indication that P(DPAEGAm-r-DBAEGAm) copolymers can be formulated into nanoparticles to control the particle disassembly and depolymerization of P(DPAEGAm-r-DBAEGAm) to occur within the pH range (pH 6.8–5.0) and time frame of endocytosis (within 24 h).

Additionally, the depolymerization assay confirmed that the amount of depolymerization could be tuned through controlling the access to the polymer end-cap. At pH 7.4, the limited depolymerization indicated that the acidic protons in solution had limited access to the DMT end-cap to trigger depolymerization. On the other hand, in cases where the PGAm polymer is solubilized in PBS at pH 7.4, the DMT end-cap is rapidly removed and extensive depolymerization quickly occurred. This result indicated that the PGAm polymer DMT end-caps were protected inside the PGAm particles at pH 7.4, which could be due to hydrophobicity, or buffering capacity of the polymers. The 4:1 PGAm particle would be expected to have the greatest side chain protonation at pH 7.4, as the particle disassembly pH was the closest to pH 7.4. This would suggest that at pH 7.4, the 4:1 PGAm particle would have the most hydrophilic core and increased migration of the polymer to the 4:1 PGAm particle and solution interface. Interestingly, the 4:1 PGAm particle showed <20% depolymerization at pH 7.4 after 1 week. On the other hand, the 0:1 PGAm particle (with the greatest side chain hydrophobicity) exhibited the greatest depolymerization (∼50%) at pH 7.4 after 1 week. As seen with the cryoTEM, the side chain composition of the PGAm polymers was shown to change the particle morphology. The increased depolymerization at pH 7.4 observed with the 0:1 PGAm particle could be the result of increased end-cap access from the changes in chain packing or the result of less buffering capacity of the pendent chain at pH 7.4.

Interestingly, previous studies that investigated pH responsive nanoparticles using the same polymer shell in combination with a nondegradable charge-shifting core showed similar particle disassembly properties to this system.34 We have also designed nanoparticles with the same shell and non-charge shifting self-immolative polymers. These systems showed no ability to disassemble.27 These previous studies suggest the particle disassembly is driven by the transition of the polymer from hydrophobic to hydrophilic not depolymerization of the core. In addition, the time scale for the depolymerization observed in this and in earlier work27 is slower than the disassembly process (2 min), which also supports the view that this system degrades through a two step degradation mechanism, including first disassembly and then depolymerization. However, the particle depolymerization experiments do suggest some depolymerization occurs while in nanoparticle for some of the systems.

To demonstrate the ability of the PGAm nanoparticles to encapsulate and release a model cargo, a HiBiT peptide was conjugated to poly[N,N-(diethylamino)ethyl methacrylate-r-N,N-(diisopropylamino)ethyl methacrylate-r-(pyridyl disulfide)ethyl methacrylate] P(DEAEMA-r-DPAEMA-r-PDSEMA) and loaded into the PGAm nanoparticles (Figure S29). Peptide cargo has therapeutic uses in vaccines and in anticancer therapy. In addition, protein complementation assays have applications in diagnostics and fundamental biochemical research.10 In particular, the NanoLuc system has been used to understand protein–protein interactions, bioluminescence imaging and understanding the intracellular trafficking of nanoparticles.10,25,26,29 The NanoLuc system is composed of two subunits, a largeBiT protein (LgBiT, 17.8 kDa) and high affinity complementary BiT (HiBiT, 1.3 kDa). Upon complementation of the HiBiT and LgBiT, the protein is able to catalyze the reaction of the substrate furimazine into furimamide, which results in the production of light measured by a luminescence reader. In a recent study, HiBiT was conjugated to P(DEAEMA-r-DPAEMA-r-PDSEMA) polymer to form a HiBiT-polymer conjugate and this conjugate was loaded into polymer nanoparticles to determine the endosomal escape efficiency of polymer nanoparticles.25

First, the HiBiT–polymer conjugate was loaded into the PGAm nanoparticles to assess the ability of the PGAm particles to encapsulate cargo and induce its release as a function of pH. To achieve this, the conjugate was loaded into the particle by diluting the conjugate solution in PBS pH 6.0 in a solution of PBS pH 8.0 and immediately followed by the P(DPAEGAm-r-DBAEGAm) and PEG-b-PDEAEMA polymer solution. The pH-responsive polymer section of the conjugate transitioned from hydrophilic to hydrophobic and began to precipitate out of solution and associate with the PGAm particles forming in solution. To confirm that the conjugate was loaded into the particle, the particles were purified by dialysis, filtered through a 0.45 μM filter, and then run through a Sepharose 6B size-exclusion column using PBS pH 8.0.

Each 1 mL elution fraction collected from the column was measured by the HiBiT luminescence assay and DLS (Figure S21). A control experiment with HiBiT peptide alone indicated that the peptide eluted in fractions 8–10 (E8–E10), and no particles were detected via DLS (Figure S21A,B). No luminescence was detected in any of the other fractions. In PBS pH 8.0, the HiBiT polymer conjugate precipitated, thus the filtered conjugate did not show luminescence in any of the Sepharose 6B elution fractions by the HiBiT luminescence assay and particles were not detected by DLS (Figure S21C,D). The HiBiT conjugate-loaded PGAm particles were observed to produce a strong luminescence signal in elution fractions and greatest count rate by DLS in elution fractions 3 and 4 (E3 and E4). Additionally, the mean diameter of the PGAm particles in E3 and E4 was ∼200 nm within the expected range for the loaded PGAm particles (Figure S21F,H,J,L). This suggested that fractions 3 and 4 (E3 and E4) just contained purified HiBiT-conjugate-loaded PGAm nanoparticles, and the HiBiT peptide was removed by the sepharose column (indicated by the low signal detected in E8 and E9). Any aggregates of the HiBiT-conjugate not incorporated into the particles were removed by the filter prior to loading onto the column. To mitigate any column variation, fractions E3 and E4 were pooled together and used as the purified HiBiT PGAm particles for all 4 systems. Additionally, in the column purification of four PGAm particles, a minor luminescence signal was detected in E8 and E9, which confirmed that a minor amount of the unconjugated and unassociated HiBiT peptide was purified in these fractions.

To quantify the loading of HiBiT conjugate into the PGAm, the HiBiT conjugate was dye labeled with Cy5, loaded into the particles, and then the Cy5 signal of the PGAm particles were measured by fluorescence spectroscopy. The loading was quantified using a fluorescence dye rather than the HiBIT assay itself as there was potential for partial or inefficient release and thus this would have underestimated the cargo (Figure S26). The different PGAm particles were found to have similar amounts of conjugate per particle with the 4:1, 2:1, 2:3, and 0:1 PGAm particles showing 0.14 ± 0.05 μg/2.5 × 109, 0.26 ± 0.06 μg/2.5 × 109, 0.17 ± 0.10 μg/2.5 × 109, and 0.24 ± 0.01 μg/2.5 × 109 particles, respectively (Figure S25).

To determine the release of the loaded HiBiT-conjugate from the PGAm particles into solution, the HiBiT luminescence assay was performed on the loaded particles across a range of pH values. The combined solution from the Sepharose 6B column fractions E3 and E4 for each PGAm particle was combined with LgBiT and the Nanoluc substrate, and the luminescence was measured (Figures 5, S23, and S24). In previous work, the amphiphilic HiBiT conjugate was observed to produce a lower luminescence signal at pH 7.4, attributed to the reduced accessibility of the HiBiT conjugate to complement LgBiT while associated with the particle.25 The data in this study shows some HiBIT is accessible, and this is why we see some signal at pH 7.4. This is due to the loading protocol that involves nanoprecipitation in the presence of the HiBIT in the aqueous media; however, we believe all HiBiT is strongly associated with the particle; otherwise, it would be removed by the column purification. In contrast, the luminescence signal greatly increased with incubation at low pH as the conjugate was released from the particle after particle disassembly and was able to complement readily with LgBiT. Therefore, the increase in the luminescence intensity provided evidence of the release of the HiBiT conjugate. In this study, the HiBiT conjugate-loaded PGAm nanoparticles were mixed with LgBiT and the NanoLuc substrate from pH 7.4 to pH 5.0 and measured by luminescence (Figure 4). The 4:1 PGAm particle provided a 6-fold increase in luminescence intensity at pH 5.0 in comparison to the pH 7.4, with the luminescence intensity rising from pH 6.8 (Figure 5A). Similarly, for the 2:1 PGAm particle, a 3-fold increase in luminescence intensity at pH 5.0 in comparison to the pH 7.4 was observed, with the luminescence intensity rising from pH 6.8 (Figure 5B). The 2:3 PGAm particle also showed an increase in luminescence intensity by a factor of 3 at pH 5.0, but the increase in luminescence began at pH 6.4. In contrast, the 0:1 PGAm particle displayed a variation in the release behavior from the rest of the PGAm particles (Figure 5D). These particles showed a small increase in luminescence (∼2-fold) at pH 6.4, but did not increase further at low pH with two of the three replicates showing no increase in luminescence intensity. In addition, the overall luminescence intensity of the pH 5.0 incubations for the 0:1 PGAm particle was much lower compared with the rest of the PGAm particles (Figure S23D). As the HiBiT-conjugate was shown to be effectively loaded in the 0:1 PGAm particle, the low luminescence reading at pH 5.0 suggests inefficient release of the conjugate. These results demonstrate the potential of PGAm particles to load and release cargo, future work will involve expanding the types of cargo to peptides and proteins with different overall charge.

Figure 5.

Figure 5

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PGAm nanoparticles show the release of the HiBiT conjugate with pH. Plot of the pH-corrected and normalized average radiance (p/s/cm2/sr) with pH for the (A) 4:1 PGAm particle, (B) 2:1 PGAm particle, (C) 2:3 PGAm particle, and (D) 0:1 PGAm particle. The luminescence was normalized to the pH 7.4 value for each run of each particle (n = 3).

Figure 4.

Figure 4

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General schematic of the release of the HiBiT conjugate from the PGAm particles. (A) Hindered access prevents the complementation of the surface-exposed HiBiT conjugate peptide with the LgBiT protein and results in muted luminescence expression. (B) Release of the HiBiT conjugate into solution enhanced accessibility for the complementation with the LgBiT protein and resulted in full luminescence expression. The LgBiT protein structure was obtained from the RSCB PDB (PDB ID: 5IBO).

To inform the dose for the SLEEQ assay, we measured the PGAm particles by a cell viability assay (Figure S27). The 2:1 PGAm particles were found to have elevated cell toxicity with <80% cell viability observed at a particle concentrations >1 × 109 particles/mL. In contrast, the 4:1, the 2:3 and the 0:1 PGAm particles showed >80% cell viability at a particle concentration <3 × 109 particles/mL. The cell viability of the 4:1 PGAm particles dropped to approximately 50% at 10 × 109 particles/mL, while the 2:3 and 0:1 maintained high viability at this particle concentration. All PGAm particles showed <20% cell viability at a particle concentration >30 × 109 particles/mL. Interestingly, the degradable nature of the PGAm polymers was not observed to reduce the toxicity of the PGAm particles in comparison to similar but nondegradable charge shifting polymeric nanoparticles.25 The heightened toxicity of the 2:1 PGAm particle suggested that more factors beyond efficient degradation affect cellular toxicity. To fully understand these factors, further investigations into the effects of nanoparticle architecture and composition on cell toxicity are ongoing. To ensure particle induced cell toxicity did not affect the SLEEQ assay results, we used a particle concentration of 2.5 × 109 particles/mL for the 4:1, the 2:3 and 0:1 PGAm particles. However, due to heightened toxicity, the 2:1 PGAm particle was excluded from the SLEEQ assay investigation.

The ability of nanoparticles to escape endosomes is critical for improving therapeutic delivery, especially for biological cargo, which is susceptible to degradation in the endo/lysosomal compartments and needs to be trafficked to other cellular compartments to be active. A number of endosomal escape mechanisms have been postulated in the literature, including the use of tailored membrane interactions based on lipid components, cell-penetrating peptides or polymers.36 However, the ability to optimize endosomal escape is still a bottleneck for many delivery systems, as highlighted by the 1–2% endosomal escape efficiency that is observed with many delivery systems.3,4,26 Another challenge in this field is the ability to accurately quantify endosomal escape. Many studies rely on qualitative assessments such as fluorescent colocalization or indirect methods such as gene knockdown or expression, which require multiple downstream processes for a change in signal to be detected. More recently, a number of direct methods to study endosomal escape have been developed, including the SLEEQ assay.32 The SLEEQ assay is a highly sensitive bioluminescent split luciferase system, which provides a quantitative measure of cytosolic delivery. In this study the SLEEQ assay was used to understand the ability for the PGAm particles to facilitate endosomal escape (Figure 6). The 4:1 PGAm particles facilitated endosomal escape of the HiBiT conjugate with a ∼4% escape efficiency. This value was calculated by ratioing the luminescence from live cells incubated with particles (measuring the amount of material delivery to the cytosol) to the luminescence of cells incubated with particles and then permeabilized with digitonin, to measure the total amount of nanoparticles associated with the cells. All PGAm particles had similar cell association. In contrast the cytosolic signal for the 4:1 system was significantly higher than those of the 2:3 and 0:1 PGAm particles, with low levels (<1%) of escape efficiency determined for the 2:3 and the 0:1 PGAm particles. The low level of escape of the 0:1 PGAm particle could be influenced by the hindered release of the HiBiT conjugate due to the higher hydrophobicity of this nanoparticle. Interestingly, the PGAm particles were less efficient at inducing endosomal escape in comparison to a similar, nondegradable system that was investigated in previous work, which showed ∼10% escape efficiency at a similar particle concentration.25 The previous work was based on nanoparticles formed by a combination of a PEG-b-PDEAEMA shell with a random copolymer core formed from poly(2-diethylamino)ethyl methacrylate (PDEAEMA) and poly(2-diisopropylamin)ethyl methacrylate (PDPAEMA). In this previous study it was shown that particles with a disassembly point of pH 6.4 were more effective at facilitating endosomal escape than a particle with a disassembly point of pH 5.0.25 The reduced escape efficiency of the PGAm particles could be due to reduced membrane interactions due to the depolymerization of the polymer chain in the endosomal compartment. Membrane interactions are an important factor in the endosomal escape of pH-responsive polymer carriers. To optimize PGAm nanoparticles for application as clinically relevant delivery systems further investigation of factors that influence membrane interaction are needed, including the rate of depolymerization and the pendent groups used.

Figure 6.

Figure 6

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4:1 PGAm nanoparticles show pronounced endosomal escape compared to the 2:3 and 0:1 PGAm nanoparticles. Plot of the (A) average radiance (p/s/cm2/sr/μg) of the total cellular association, (B) the cytosolic luminescence signal (p/s/cm2/sr/μg), and (C) the escape efficiency of the SLEEQ assay for the 4:1 PGAm particles (red), 2:3 PGAm particles (yellow), and 0:1 PGAm particles (gray) incubated for 4 h at 2.5 × 109 particles/mL in HEK293-LSA cells. The average radiance of (A) and (B) were background-corrected and normalized to the mass of conjugate dosed to each well. Data represents mean ± SEM, n = 3. Two-tailed unpaired t test was used to analyze the data, ns denotes not significant, *p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001.

Conclusion

In conclusion, two-stage pH responsive nanoparticles based on charge shifting and pH responsive self-immolative polymer P(DPAEGAm-r-DBAEGAm) were developed. The pH of particle disassembly was tuned from pH 7.0 to pH 5.0 through the ratio of diisopropylamino to dibutylamino substituents on the pendent tertiary amine. The PGAm polymer was observed to extensively depolymerize (60–80%) below the particle disassembly pH after 2 h, compared to <10% depolymerization observed at pH 7.4 over the same period. The PGAm particles showed reasonable stability at pH 7.4 (<20–50% depolymerization) after 1 week, which made them more attractive for application as drug delivery systems. A model peptide (HiBiT) conjugate was loaded into the PGAm particles, and found to be efficiently released from the 4:1, 2:1, and 2:3 particles, however, the release was ineffective from the 0:1 PGAm particle, likely due to increased hydrophobicity of this system. The effective delivery of cargo was investigated by studying the endosomal escape of the cargo using the SLEEQ assay. Interestingly, the 4:1 PGAm particle was observed to induce ∼4% endosomal escape efficiency, which is higher than nondegradable PDEAEMA/PDPAEMA carriers that have been investigated by this assay previously. Thus the 4:1 PGAm system is a promising candidate for further investigation for intracellular drug delivery and other pH-responsive drug delivery applications.

Acknowledgments

G.S. and E.R.G. would like to acknowledge funding from the Australian Research Council (DP180100844). E.R.G. would like to acknowledge the Canada Research Chairs program (CRC-2020-00101).

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.biomac.3c00630.

  • Synthesis and characterization of all PGAm polymers and precursor amines, including NMR and GPC characterization. Nanoparticle preparation and characterization methods including NanoSight, TEM, NMR depolymerization studies, loading of HiBiT, and cell viability assays (PDF)

The authors declare no competing financial interest.

Supplementary Material

bm3c00630_si_001.pdf (2.3MB, pdf)

References

  1. Farokhzad O. C.; Langer R. Impact of Nanotechnology on Drug Delivery. ACS Nano 2009, 3, 16–20. 10.1021/nn900002m. [DOI] [PubMed] [Google Scholar]
  2. Stewart M. P.; Sharei A.; Ding X.; Sahay G.; Langer R.; Jensen K. F. In vitro and ex vivo strategies for intracellular delivery. Nature 2016, 538, 183–192. 10.1038/nature19764. [DOI] [PubMed] [Google Scholar]
  3. Wittrup A.; Ai A.; Liu X.; Hamar P.; Trifonova R.; Charisse K.; Manoharan M.; Kirchhausen T.; Lieberman J. Visualizing lipid-formulated siRNA release from endosomes and target gene knockdown. Nat. Biotechnol. 2015, 33, 870–876. 10.1038/nbt.3298. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Gilleron J.; Querbes W.; Zeigerer A.; Borodovsky A.; Marsico G.; Schubert U.; Manygoats K.; Seifert S.; Andree C.; Stöter M.; Epstein-Barash H.; Zhang L.; Koteliansky V.; Fitzgerald K.; Fava E.; Bickle M.; Kalaidzidis Y.; Akinc A.; Maier M.; Zerial M. Image-based analysis of lipid nanoparticle–mediated siRNA delivery, intracellular trafficking and endosomal escape. Nat. Biotechnol. 2013, 31, 638–646. 10.1038/nbt.2612. [DOI] [PubMed] [Google Scholar]
  5. Mao T.; Israelow B.; Peña-Hernández M. A.; Suberi A.; Zhou L.; Luyten S.; Reschke M.; Dong H.; Homer R. J.; Saltzman W. M.; Iwasaki A. Unadjuvanted intranasal spike vaccine elicits protective mucosal immunity against sarbecoviruses. Science 2022, 378, eabo2523 10.1126/science.abo2523. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Ndeupen S.; Qin Z.; Jacobsen S.; Bouteau A.; Estanbouli H.; Igyártó B. Z. The mRNA-LNP platform’s lipid nanoparticle component used in preclinical vaccine studies is highly inflammatory. iScience 2021, 24, 103479. 10.1016/j.isci.2021.103479. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Such G. K.; Yan Y.; Johnston A. P. R.; Gunawan S. T.; Caruso F. Interfacing Materials Science and Biology for Drug Carrier Design. Adv. Mater. 2015, 27, 2278–2297. 10.1002/adma.201405084. [DOI] [PubMed] [Google Scholar]
  8. Neri D.; Supuran C. T. Interfering with pH regulation in tumours as a therapeutic strategy. Nat. Rev. Drug Discovery 2011, 10, 767–777. 10.1038/nrd3554. [DOI] [PubMed] [Google Scholar]
  9. Feng L.; Dong Z.; Tao D.; Zhang Y.; Liu Z. The acidic tumor microenvironment: a target for smart cancer nano-theranostics. National Sci. Rev. 2018, 5, 269–286. 10.1093/nsr/nwx062. [DOI] [Google Scholar]
  10. Deirram N.; Zhang C.; Kermaniyan S. S.; Johnston A. P. R.; Such G. K. pH-Responsive Polymer Nanoparticles for Drug Delivery. Macromol. Rapid Commun. 2019, 40, 1800917. 10.1002/marc.201800917. [DOI] [PubMed] [Google Scholar]
  11. Rennick J. J.; Nowell C. J.; Pouton C. W.; Johnston A. P. R. Resolving subcellular pH with a quantitative fluorescent lifetime biosensor. Nat. Commun. 2022, 13, 6023. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Englert C.; Brendel J. C.; Majdanski T. C.; Yildirim T.; Schubert S.; Gottschaldt M.; Windhab N.; Schubert U. S. Pharmapolymers in the 21st century: Synthetic polymers in drug delivery applications. Prog. Polym. Sci. 2018, 87, 107–164. 10.1016/j.progpolymsci.2018.07.005. [DOI] [Google Scholar]
  13. Zhou K.; Wang Y.; Huang X.; Luby-Phelps K.; Sumer B. D.; Gao J. Tunable, Ultrasensitive pH-Responsive Nanoparticles Targeting Specific Endocytic Organelles in Living Cells. Angew. Chem., Int. Ed. 2011, 50, 6109–6114. 10.1002/anie.201100884. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Vaidyanathan S.; Orr B. G.; Banaszak Holl M. M. Role of Cell Membrane–Vector Interactions in Successful Gene Delivery. Acc. Chem. Res. 2016, 49, 1486–1493. 10.1021/acs.accounts.6b00200. [DOI] [PubMed] [Google Scholar]
  15. Vaidyanathan S.; Chen J.; Orr B. G.; Banaszak Holl M. M. Cationic Polymer Intercalation into the Lipid Membrane Enables Intact Polyplex DNA Escape from Endosomes for Gene Delivery. Mol. Pharmaceutics 2016, 13, 1967–1978. 10.1021/acs.molpharmaceut.6b00139. [DOI] [PubMed] [Google Scholar]
  16. Yardley R. E.; Kenaree A. R.; Gillies E. R. Triggering Depolymerization: Progress and Opportunities for Self-Immolative Polymers. Macromolecules 2019, 52, 6342–6360. 10.1021/acs.macromol.9b00965. [DOI] [Google Scholar]
  17. Lee O. P.; Lopez Hernandez H.; Moore J. S. Tunable Thermal Degradation of Poly(vinyl butyl carbonate sulfone)s via Side-Chain Branching. ACS Macro Lett. 2015, 4, 665–668. 10.1021/acsmacrolett.5b00234. [DOI] [PubMed] [Google Scholar]
  18. DiLauro A. M.; Lewis G. G.; Phillips S. T. Self-Immolative Poly (4, 5-dichlorophthalaldehyde) and its Applications in Multi-Stimuli-Responsive Macroscopic Plastics. Angew. Chem., Int. Ed. 2015, 127, 6298–6303. 10.1002/ange.201501320. [DOI] [PubMed] [Google Scholar]
  19. Fan B.; Trant J. F.; Gillies E. R. End-Capping Strategies for Triggering End-to-End Depolymerization of Polyglyoxylates. Macromolecules 2016, 49, 9309–9319. 10.1021/acs.macromol.6b02320. [DOI] [Google Scholar]
  20. Roth M. E.; Green O.; Gnaim S.; Shabat D. Dendritic, Oligomeric, and Polymeric Self-Immolative Molecular Amplification. Chem. Rev. 2016, 116, 1309–1352. 10.1021/acs.chemrev.5b00372. [DOI] [PubMed] [Google Scholar]
  21. Gopin A.; Ebner S.; Attali B.; Shabat D. Enzymatic Activation of Second-Generation Dendritic Prodrugs: Conjugation of Self-Immolative Dendrimers with Poly(ethylene glycol) via Click Chemistry. Bioconjugate Chem. 2006, 17, 1432–1440. 10.1021/bc060180n. [DOI] [PubMed] [Google Scholar]
  22. Fomina N.; McFearin C.; Sermsakdi M.; Edigin O.; Almutairi A. UV and Near-IR Triggered Release from Polymeric Nanoparticles. J. Am. Chem. Soc. 2010, 132, 9540–9542. 10.1021/ja102595j. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Fan B.; Gillies E. R. Poly(ethyl glyoxylate)-Poly(ethylene oxide) Nanoparticles: Stimuli-Responsive Drug Release via End-to-End Polyglyoxylate Depolymerization. Mol. Pharmaceutics 2017, 14, 2548–2559. 10.1021/acs.molpharmaceut.7b00030. [DOI] [PubMed] [Google Scholar]
  24. McKinlay C. J.; Benner N. L.; Haabeth O. A.; Waymouth R. M.; Wender P. A. Enhanced mRNA delivery into lymphocytes enabled by lipid-varied libraries of charge-altering releasable transporters. Proc. Natl. Acad. Sci. U.S.A. 2018, 115, E5859–E5866. 10.1073/pnas.1805358115. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Benner N. L.; McClellan R. L.; Turlington C. R.; Haabeth O. A. W.; Waymouth R. M.; Wender P. A. Oligo(serine ester) Charge-Altering Releasable Transporters: Organocatalytic Ring-Opening Polymerization and their Use for in Vitro and in Vivo mRNA Delivery. J. Am. Chem. Soc. 2019, 141, 8416–8421. 10.1021/jacs.9b03154. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Sirianni Q. E. A.; Rabiee Kenaree A.; Gillies E. R. Polyglyoxylamides: Tuning Structure and Properties of Self-Immolative Polymers. Macromolecules 2019, 52, 262–270. 10.1021/acs.macromol.8b02616. [DOI] [Google Scholar]
  27. Zhang C.; Kermaniyan S.; Smith S. A.; Gillies E. R.; Such G. K. Acid-Responsive Poly(glyoxylate) Self-Immolative Star Polymers. Biomacromolecules 2021, 22, 3892–3900. 10.1021/acs.biomac.1c00694. [DOI] [PubMed] [Google Scholar]
  28. McBride R. A.; Gillies E. R. Kinetics of Self-Immolative Degradation in a Linear Polymeric System: Demonstrating the Effect of Chain Length. Macromolecules 2013, 46, 5157–5166. 10.1021/ma4009753. [DOI] [Google Scholar]
  29. Fan B.; Trant J. F.; Wong A. D.; Gillies E. R. Polyglyoxylates: A Versatile Class of Triggerable Self-Immolative Polymers from Readily Accessible Monomers. J. Am. Chem. Soc. 2014, 136, 10116–10123. 10.1021/ja504727u. [DOI] [PubMed] [Google Scholar]
  30. Yeung K.; Kim H.; Mohapatra H.; Phillips S. T. Surface-Accessible Detection Units in Self-Immolative Polymers Enable Translation of Selective Molecular Detection Events into Amplified Responses in Macroscopic, Solid-State Plastics. J. Am. Chem. Soc. 2015, 137, 5324–5327. 10.1021/jacs.5b02799. [DOI] [PubMed] [Google Scholar]
  31. Beach M. A.; Teo S. L. Y.; Chen M. Z.; Smith S. A.; Pouton C. W.; Johnston A. P. R.; Such G. K. Quantifying the Endosomal Escape of pH-Responsive Nanoparticles Using the Split Luciferase Endosomal Escape Quantification Assay. ACS Appl. Mater. Interfaces 2022, 14, 3653–3661. 10.1021/acsami.1c18359. [DOI] [PubMed] [Google Scholar]
  32. Teo S. L. Y.; Rennick J. J.; Yuen D.; Al-Wassiti H.; Johnston A. P. R.; Pouton C. W. Unravelling cytosolic delivery of cell penetrating peptides with a quantitative endosomal escape assay. Nat. Commun. 2021, 12, 3721. 10.1038/s41467-021-23997-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Wong A. S. M.; Mann S. K.; Czuba E.; Sahut A.; Liu H.; Suekama T. C.; Bickerton T.; Johnston A. P. R.; Such G. K. Self-assembling dual component nanoparticles with endosomal escape capability. Soft Matter 2015, 11, 2993–3002. 10.1039/C5SM00082C. [DOI] [PubMed] [Google Scholar]
  34. Kongkatigumjorn N.; Smith S. A.; Chen M.; Fang K.; Yang S.; Gillies E. R.; Johnston A. P. R.; Such G. K. Controlling Endosomal Escape Using pH-Responsive Nanoparticles with Tunable Disassembly. ACS Appl. Nano Mater. 2018, 1, 3164–3173. 10.1021/acsanm.8b00338. [DOI] [Google Scholar]
  35. Sirianni Q. E.; Liang X.; Such G. K.; Gillies E. R. Polyglyoxylamides with a pH-Mediated Solubility and Depolymerization Switch. Macromolecules 2021, 54, 10547–10556. 10.1021/acs.macromol.1c01796. [DOI] [Google Scholar]
  36. Mollé L. M.; Smyth C. H.; Yuen D.; Johnston A. P. Nanoparticles for vaccine and gene therapy: Overcoming the barriers to nucleic acid delivery. WIREs Nanomed. Nanobiotechnol. 2022, 14, e1809 10.1002/wnan.1809. [DOI] [PMC free article] [PubMed] [Google Scholar]

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