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. 2026 Jun 2;2(9):693–705. doi: 10.1021/polymscitech.6c00029

Hydrolytically Degradable Polyethylene Glycol Bottlebrush Polymers Address the Stability-Clearance Trade-Off for Drug Delivery

Yixing Wen †,‡, Na Zhang ‡,§, Simin Sun †, Chen Yang †,‡, Xingdi Cheng †,‡, Shiwei Mi †,‡, Guizhi Shi ‡,∥,⊥, Xin Huang #,*, Wei You ‡,§,*, Xueguang Lu †,‡,*
PMCID: PMC13614148  PMID: 42799416

Abstract

Long-circulating polyethylene glycol bottlebrush polymers (BrushPEG) are attractive drug carriers, but their nondegradable backbones cause persistent tissue retention, raising concerns about long-term safety and clinical translation. Here, we report a hydrolytically degradable PEG bottlebrush polymer (D-BrushPEG) created by incorporating 7-oxa-2,3-diazanorbornene (ODAN) units into the backbone. D-BrushPEG retains prolonged circulation (elimination half-life ≈24.7 h, compared with ≈0.78 h for a small-molecule dye) while undergoing controlled hydrolysis that greatly improves systemic and tissue clearance. More than 99% of the circulating signal is removed within 13 days, accompanied by a near-complete loss of liver and skin fluorescence. In contrast, a nondegradable BrushPEG analog exhibits slower plasma elimination (∼5% remaining at day 13) and persistent accumulation in the liver and skin for over one month. When conjugated with gemcitabine (GEM) through a reduction-responsive self-immolative linker, D-BrushPEG demonstrated comparable antitumor efficacy to the nondegradable BrushPEG-GEM formulation. Systemic evaluation indicates comparable hepatic and renal safety profiles for both polymers, with only mild renal changes observed, while skin irritation is reduced for D-BrushPEG relative to BrushPEG. These results demonstrate that controlled backbone hydrolysis allows PEG bottlebrush polymers to balance long circulation with efficient clearance, reducing tissue persistence and the risk of side effects.

Keywords: bottlebrush polymer, degradable polymer, polyethylene glycol, drug delivery, nanomedicine

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Introduction

Cancer remains one of the leading causes of death worldwide, responsible for nearly 10 million deaths annually according to the International Agency for Research on Cancer. Despite remarkable advances in targeted therapies, immunotherapies, and combination treatment regimens, chemotherapy remains a major first-line therapeutic option for many cancers. − Nonetheless, its effectiveness is limited by poor pharmacokinetics, systemic toxicity, and a lack of selectivity. Most small-molecule chemotherapeutics suffer from rapid clearance, nonspecific biodistribution, and narrow therapeutic windows, leading to dose-limiting side effects that compromise patient outcomes. , Nanomedicine has emerged as a powerful approach to address these challenges, with polymeric micelles, liposomes, and albumin-based carriers already approved for clinical use. , These nanocarrier systems enhance drug solubility and extend plasma half-life, leading to improved tumor accumulation. , However, their therapeutic impact remains limited by insufficient stability and short systemic retention, both of which are key determinants of tumor delivery efficiency. These limitations highlight the need for new delivery systems that can achieve high payload capacity and extended blood circulation. ,

Polyethylene glycol bottlebrush polymers (BrushPEG) have recently emerged as a promising nanocarrier platform for chemotherapeutic delivery. − Their densely grafted side-chain architecture gives rise to discrete, single-molecule nanoparticles in which the backbone, side chains, and conjugated cargo are all covalently integrated. − This design confers exceptional structural stability and creates a multifunctional scaffold bearing numerous pendant arms radiating from a central backbone. Such an arrangement enables the high-density and versatile conjugation of therapeutic payloads, ranging from small-molecule drugs to nucleic acids, while also accommodating targeting ligands or other functional moieties within a single construct. − When PEG is employed as the side chain, BrushPEG further benefit from reduced protein adsorption, prolonged circulation half-life, and improved tissue penetration, collectively enhancing tumor accumulation. − Recent advances have substantiated these advantages across multiple therapeutic contexts. − BrushPEG carrying chemotherapeutics have achieved ultrahigh loading densities and potent tumor inhibition in vivo, while nucleic acid–conjugated BrushPEG have demonstrated improved pharmacokinetics and enhanced gene-silencing efficacy. ,

Despite these advantages, the exceptional stability of BrushPEG also introduces a critical liability: most BrushPEG possess nondegradable backbones that prevent renal clearance, leading to long-term retention in the liver, spleen, and skin. Such persistence raises safety concerns, as prolonged nanoparticle accumulation in organs has been associated with chronic inflammatory and fibrotic responses in animal studies. Recent efforts have therefore focused on introducing degradable motifs such as silyl ethers, esters, , phosphoesters, cyclooctanes, , and disulfides − into BrushPEG backbones. However, these chemistries typically rely on specific triggerssuch as acidic pH, enzymatic activity, or reductive environmentsto initiate backbone cleavage. As these physiological triggers are inherently heterogeneous and vary significantly across tissues, disease states, and individuals, the resulting degradation behavior can be difficult to predict and control. Therefore, there remains a need for degradable brush polymer systems that retain the pharmacokinetic advantages of PEGylation while enabling predictable, physiologically driven clearance.

Here, we report a hydrolytically degradable BrushPEG (D-BrushPEG) that addresses this stability-clearance paradox. The design integrates 7-oxa-2,3-diazanorbornene (ODAN), a ring-opening metathesis polymerization (ROMP)-compatible monomer that undergoes spontaneous hydrolysis under physiological conditions, to introduce uniformly distributed degradable sites along the polymer backbone. , The resulting D-BrushPEG retains the hallmark properties of conventional BrushPEGmolecular precision, high drug-loading density, and extended blood circulationyet undergoes backbone cleavage that facilitates timely systemic clearance (Figure A). Using gemcitabine (GEM) as a model payload, we show that D-BrushPEG achieves comparable tumor inhibition and reduced long-term retention compared with a nondegradable BrushPEG analog.

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Comparative schematic diagram of D-BrushPEG and BrushPEG. (A) The schematic diagram delineates the in vivo long-circulation and tumor-suppression capabilities of D-BrushPEG and BrushPEG systems, and the respective in vivo biodegradation/clearance or organ accumulation profiles of D-BrushPEG versus BrushPEG. (B) The synthetic routes and degradation of D-BrushPEG and BrushPEG.

Results and Discussion

Synthesis and Characterization of D-BrushPEG and BrushPEG

The bottlebrush polymers were synthesized using a graft-onto strategy (Figure B). The degradable backbone (DPB, poly­(NB-NHS-ran-ODAN)) was synthesized through ROMP of norbornene-NHS (NB-NHS) and ODAN. Due to their comparable reactivity, a feed ratio of 3:2 was selected to ensure uniform distribution of the degradable ODAN along the polymer chain. The polymer was designed to contain approximately 100 NHS ester groups, providing reactive sites for subsequent conjugation with PEG or therapeutic molecules.

1H NMR spectroscopy confirmed the successful synthesis of DPB, showing characteristic signals at δ = 6.77–4.96, 4.23, 1.28 ppm corresponding to ODAN incorporation (Figure S1–S5). 1H NMR analysis shows that the ODAN-to-norbornene–NHS ratio is approximately 1.42:1, consistent with the monomer feed ratio, confirming successful incorporation of ODAN units into the polymer backbone (Figure S5). The random distribution of ODAN and NB–NHS units in the polymer backbone is supported by their comparable copolymerization reactivity ratios reported previously, which favor statistically random incorporation during ROMP polymerization. As a nondegradable control, poly­(NB-NHS) (NPB) was synthesized by homopolymerization of NB-NHS. Both NPB (Mn = 19.0 kDa, PDI = 1.24) and DPB (Mn = 43.1 kDa, PDI = 1.08) displayed narrow molecular weight distributions as determined by gel permeation chromatography (GPC). The higher molecular weight of DPB, as compared to NPB, was consistent with the incorporation of ODAN units (Figure A).

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Preparation and characterizations of D-BrushPEG and BrushPEG. (A) GPC chromatograms of DPB, NPB, D-BrushPEG, and BrushPEG. (B) Ninhydrin-stained thin-layer chromatography (TLC) of reaction mixtures containing DPB or NPB reacted with increasing molar ratios of PEG–NH2. The labeled numbers represent the molar ratio of PEG to NHS corresponding to the adjacent spots; unmarked spots correspond to stock PEG solution and its 1% dilution. (C) Intensity-weighted hydrodynamic size distributions and (D) zeta potentials of D-BrushPEG and BrushPEG as measured by DLS (n = 3 technical replicates). (E) Representative TEM images and (F) corresponding particle size analysis of D-BrushPEG and BrushPEG (100 particles counted per sample), scale bar is 100 nm. Data are shown as mean ± standard deviation (SD).

Amine-terminated PEG5k were then grafted onto the polymer through amidation. The optimal grafting ratio was determined by reacting NPB with amine-modified PEG at varying molar ratios, followed by ninhydrin assay to quantify residual NH2 on PEG after overnight incubation. Quantitative conjugation was achieved when the PEG feed ratio was ≤70% of the NHS content. Further increases in the PEG resulted in detectable free amino groups. The remaining NHS sites were likely inaccessible by PEG due to steric hindrance (Figure B).

To preserve additional NHS sites for subsequent modification while ensuring quantitative PEGylation, the brush was designed to incorporate ∼50 PEG5k chains per backbone, yielding the nondegradable BrushPEG and degradable D-BrushPEG. 1H NMR analysis indicates ∼55 and ∼60 PEG chains per polymer for D-BrushPEG and BrushPEG, respectively (Figure S6 and S7), consistent with high grafting efficiency. Following PEG grafting, the molecular weights of BrushPEG and D-BrushPEG increased to 69.8 kDa (PDI = 1.08) and 110.2 kDa (PDI = 1.09), respectively, consistent with the expected incorporation of PEG chains (Figure A). This modification markedly improved water solubility. These data demonstrate the successful synthesis of BrushPEG and D-BrushPEG.

Previous studies have shown that sufficiently high-molecular-weight BrushPEG behave as discrete unimolecular nanoparticles. Dynamic light scattering (DLS) revealed hydrodynamic diameters of 44.4 nm for D-BrushPEG and 33.3 nm for BrushPEG, which are consistent with their relative molecular weights (Figure C). Both brush polymers exhibited near-neutral surface potentials (Figure D). Transmission electron microscopy (TEM) analysis showed spherical features for both brushes with mean diameters of 23.3 nm for D-BrushPEG and 17.2 nm for BrushPEG (Figure E and F), consistent with previously reported morphologies of PEGylated brush polymers under negative staining conditions. ,,,,

Degradation and Cellular Uptake of D-BrushPEG and BrushPEG

In aqueous environments, the ODAN unit undergoes hydrolysis by a 1,3-dipolar cycloreversion mechanism, which has been studied in our previous work. The cleavage produces polymer fragments bearing aldehyde-terminated chain ends, along with corresponding hydrazide/amine-containing counterparts (Figure B). Because the ODAN motif is incorporated directly into the polymer backbone, this transformation results in progressive chain cleavage and reduction in molecular weight over time.

To experimentally verify this hydrolytic degradation behavior under physiologically relevant conditions, D-BrushPEG and BrushPEG were incubated in phosphate-buffered saline (PBS, pH 7.4) at 37 °C and analyzed at predetermined time points by DLS and GPC. Notably, during the degradation of D-BrushPEG, the particle size exhibited a trend of initial decrease followed by an increase (Figure A). Clearly, the size reduction observed at 8 h results from backbone cleavage that generates smaller fragments, and at this stage the degradation products are still predominantly PEG-rich particle fragments. However, with further degradation, DLS analysis revealed the emergence of a population with larger sizes, which was unexpected. The underlying mechanism is not yet fully understood. We speculate that, during degradation, the PEG content on each fragment gradually decreases. Given that neither the ODAN units nor the aldehyde groups generated upon degradation are strongly hydrophilic, once the PEG shielding is reduced, those fragments enriched in these relatively less hydrophilic moieties may aggregate, resulting in the formation of larger particle populations. In contrast, BrushPEG remained stable with negligible changes in size (Figure B).

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Degradation and cellular uptake properties of brush polymers. (A) Hydrodynamic size distributions of D-BrushPEG and (B) BrushPEG after incubation in PBS (pH 7.4) at 37 °C (n = 3 technical replicates). (C) GPC chromatograms for D-BrushPEG and (D) BrushPEG after incubation in PBS at 37 °C. (E) Schematic illustration showing the degradation of D-BrushPEG. (F) Schematic for the synthesis of Cy5-labeled brushes. (G) Flow cytometry analysis of Cy5 mean fluorescence intensity (MFI) in DC2.4 cells treated with Cy5-labeled D-BrushPEG or BrushPEG (n = 3 technical replicates). (H) Schematic of endocytic pathways targeted by inhibitors. (I) Normalized cellular uptake of Cy5-labeled D-BrushPEG and BrushPEG in DC2.4 cells treated with various endocytic pathway inhibitors (n = 3 technical replicates). Data are shown as mean ± standard error of the mean (SEM) in (A, B) and as mean ± SD in (G, I). Statistical significance was calculated by one-way analysis of variance (ANOVA) and Tukey’s multiple comparisons test in (I). ****P < 0.0001.

GPC analysis showed a marked reduction in D-BrushPEG molecular weight after incubation (Figure C). BrushPEG exhibited a minor rightward shift at early time points, which may be associated with changes following hydrolysis of residual NHS esters. In addition, the formation of carboxyl groups may introduce interactions with the column media, potentially contributing to the observed delay in elution time (Figure D). The GPC profiles of D-BrushPEG at 24 and 48 h were nearly identical, indicating that degradation was nearly complete within 24 h. The results from both DLS and GPC confirm the hydrolytic degradability of D-BrushPEG in aqueous solution at neutral pH (Figure E). To avoid unnecessary degradation, D-BrushPEG was lyophilized and then stored at −20 °C.

We next assessed cellular uptake of D-BrushPEG and BrushPEG. For quantitative analysis, amine-modified Cyanine5 (Cy5) dye was conjugated to D-BrushPEG and BrushPEG via amidation (Figure F). Cy5-modified brushes were incubated with DC2.4 cells and analyzed by flow cytometry. D-BrushPEG exhibited substantially higher cellular uptake than BrushPEG, with the enhancement becoming more pronounced over time (Figure G).

To determine whether this enhancement originated from differences in endocytic pathways, DC2.4 cells were pretreated with inhibitors of caveolae-mediated endocytosis (methyl-β-cyclodextrin, MβCD), clathrin-mediated endocytosis (chlorpromazine, CPZ), or macropinocytosis (wortmannin, Wort), or incubated at 4 °C to suppress energy-dependent uptake (Figure H). , Both polymers displayed markedly reduced uptake at 4 °C, confirming energy-dependent internalization. CPZ treatment significantly inhibited the uptake of both D-BrushPEG and BrushPEG, indicating a predominant reliance on clathrin-mediated endocytosis (Figure I).

The similarity in uptake mechanisms suggests that the differences in cellular internalization do not arise from distinct endocytic pathways. Instead, the enhanced uptake of D-BrushPEG is likely related to degradation-induced changes in its physicochemical properties. As degradation proceeds, increased chain flexibility and the formation of fragments with reduced PEG shielding may enhance interactions with the cell membrane, thereby promoting cellular internalization.

Pharmacokinetics and Biodistribution of D-BrushPEG and BrushPEG

PEG bottlebrush polymers are known to exhibit prolonged blood circulation, reflecting their inherent stealth characteristics derived from dense PEG functionalization. To study the pharmacokinetics and biodistribution, Cy5-labeled D-BrushPEG and BrushPEG were intravenously administered in female ICR mice. Blood samples were collected at different time points for Cy5 quantification. Both brush polymers exhibited a markedly longer circulation time than free Cy5 (Figure A and B). Pharmacokinetic analysis revealed elimination half-lives of 24.7 h for D-BrushPEG and 40.2 h for BrushPEG, whereas free Cy5 dye was rapidly cleared with a half-life of only 0.78 h (Figure C), demonstrating the role of PEGylation in prolonging brush polymers’ circulation time.

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Prolonged systemic circulation and tumor-targeting ability of brush polymers. (A) Plasma pharmacokinetic profiles of free Cy5, Cy5-labeled D-BrushPEG, and Cy5-labeled BrushPEG following intravenous injection (10 nmol Cy5 per mouse, n = 3 biologically independent samples). (B) AUCs and (C) elimination half-lives of free Cy5, Cy5-labeled D-BrushPEG, and Cy5-labeled BrushPEG. (D) Schematic showing the enhanced tumor accumulation of brush polymers. (E) Representative IVIS images and (F, G) quantifications of Cy5 fluorescence in Balb/c-nu mice bearing subcutaneous HeLa xenografts (10 nmol Cy5 per mouse, n = 3 biologically independent samples). (H) Quantification of residual Cy5 fluorescence in plasma on day 13 post-injection. (I) Ex vivo fluorescence images and (J) quantitative analysis of Cy5 signal in major organs on day 13 post-injection. H, heart; Li, liver; Sp, spleen; Lu, lung; K, kidney; T, tumor; Sk, skin. Data are shown as mean ± SD. Statistical significance was calculated by one-way ANOVA and Tukey’s multiple comparisons test in (B, C, H); two-way ANOVA with Tukey’s multiple comparisons test in (F, G, J). ns, not significant; *P < 0.05; **P < 0.01; ****P < 0.0001.

Although nanoparticle size can influence cellular uptake and pharmacokinetics, D-BrushPEG (∼44 nm) and BrushPEG (∼33 nm) fall within a relatively narrow size range and share similar PEG surface characteristics, which are expected to minimize size-dependent differences. , Instead, the distinct degradability of D-BrushPEG, enabling fragmentation into smaller species in vivo, is likely the primary factor contributing to its altered cellular uptake and pharmacokinetic behavior.

Prolonged circulation half-life generally enables nanoparticles to achieve enhanced accumulation at the disease site. In degradable systems, polymers can accumulate in tumors and subsequently undergo clearance through controlled backbone degradation (Figure D). To evaluate the in vivo behavior of the polymers, a subcutaneous HeLa xenograft model was established in Balb/c-nu mice, and Cy5-labeled polymers were administered intravenously. In vivo imaging system (IVIS) imaging showed that both brush polymers accumulated in tumors to a much greater extent than free Cy5 (Figure E). D-BrushPEG exhibited a circulation half-life of 24.7 h, indicating that the polymer remains sufficiently stable in vivo to enable prolonged systemic exposure and tumor accumulation. Separately, the in vitro degradation study demonstrated that D-BrushPEG is hydrolytically labile under physiological conditions, supporting its potential for subsequent fragmentation and clearance. Together, these results suggest that D-BrushPEG achieves a favorable balance between in vivo persistence and degradability, although the in vitro degradation time scale should not be interpreted as a direct surrogate for its in vivo pharmacokinetics.

On day 13, the whole-body Cy5 fluorescence signal of D-BrushPEG and BrushPEG was 32.4-fold and 185.2-fold higher than that of free Cy5, respectively, while tumor fluorescence was 70.8-fold and 468.7-fold higher (Figure F and G). Analysis of the area under the curve (AUC) showed a similar trend: whole-body fluorescence AUC for D-BrushPEG and BrushPEG was 27.0- and 77.1-fold greater than that of free Cy5, respectively, and tumor fluorescence AUC was 71.8- and 229.4-fold greater (Figure S8 and S9).

Fluorescence analysis of blood samples showed that D-BrushPEG was >99.4% cleared by day 13, whereas BrushPEG remained detectable at ∼5.3% (Figure H). Ex vivo imaging further confirmed predominant accumulation of both polymers in the liver, tumor, and skindistribution patterns typical of PEG brush polymers (Figure I and J). These data reveal that BrushPEG exhibits stronger and more sustained tumor retention, while D-BrushPEG achieves efficient tumor delivery coupled with timely systemic elimination.

In Vivo Anti-Tumor Efficacy of GEM-Loaded D-BrushPEG and BrushPEG

To evaluate therapeutic performance, GEM was selected as a model chemotherapeutic owing to its clinical relevance. To ensure the release of intact GEM and preserve its pharmacological activity, a reduction-responsive self-immolative linker was introduced. ,, The linker contains a terminal amine group that forms an amide bond with the NHS-functionalized polymer backbone (Figure A). Successful synthesis of linker-GEM was demonstrated through NMR and MS (Figure S10–S26). Under the reductive endosomal environment, the linker cleaves to release free GEM (Figure B). The modified GEM was covalently attached to the polymer backbone at a substitution ratio of 20%. The completion of the reaction was confirmed by thin-layer chromatography (TLC) (Figure C). Upon incubation with glutathione (GSH), D-BrushPEG and BrushPEG both efficiently released free GEM as determined by high-performance liquid chromatography (HPLC). In contrast, polymers without GSH treatment showed negligible GEM release, suggesting the reduction-responsive release of GEM (Figure D and S27). In vitro cytotoxicity assays showed that both GEM-loaded brushes effectively inhibited HeLa cell proliferation. GEM-modified D-BrushPEG and BrushPEG displayed comparable cytotoxicity at low and medium concentrations, whereas D-BrushPEG produced stronger cell killing at higher doses (Figure E). This enhancement is consistent with more efficient cellular uptake facilitated by the degradable backbone (Figure G).

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Synthesis and antitumor evaluation of GEM-loaded brush polymers. (A) Schematic of GEM modified with a self-immolative linker grafted onto the polymer. (B) Schematic of GEM release from the polymer under reductive conditions. (C) TLC analysis of GEM-conjugated polymer by ninhydrin staining. (D) GSH-dependent GEM release kinetics from polymers (n = 3 technical replicates). (E) Viability of HeLa cells treated with free GEM, GEM-loaded D-BrushPEG, and GEM loaded BrushPEG (n = 4 technical replicates). (F) Treatment regimen for subcutaneous HeLa tumor-bearing Balb/c-nu mice (n = 5 biologically independent samples for each group). (G) Tumor volume changes over time. (H) Photographs and (I) weight of isolated tumors 7 days after the last administration. (J) Changes in body weight during treatment. Data are shown as mean ± SD in (D, E, I, J) and as mean ± SEM in (G). Statistical significance was calculated by two-way ANOVA with Tukey’s multiple comparisons test in (G) and by one-way ANOVA with Tukey’s multiple comparisons test in (I). *P < 0.05; ****P < 0.0001.

We next evaluated the in vivo therapeutic efficacy of D-BrushPEG in a subcutaneous HeLa xenograft model (Figure F). Both GEM-conjugated D-BrushPEG and BrushPEG outperformed free GEM in tumor inhibition (Figure G), likely due to enhanced tumor accumulation. Both D-BrushPEG–GEM and BrushPEG–GEM produced comparable tumor growth inhibition under the tested conditions. The ex vivo data from tumors collected on day 7 posttreatment demonstrated a trend consistent with the in vivo experimental results (Figure H), with tumor weight in the D-BrushPEG-treated group reduced to ∼40.3% of the PBS group (Figure I).

While D-BrushPEG exhibited a lower tumor accumulation ratio and shorter retention time compared to BrushPEG, it still demonstrated comparable antitumor efficacy. In addition to the enhanced cellular uptake, we hypothesized that the degradation products of D-BrushPEG may contribute additional antitumor activity. To test this, D-BrushPEG without GEM conjugation was co-incubated with HeLa and SKBR-3 cells. D-BrushPEG reduced cell viability at higher concentrations, whereas BrushPEG showed minimal cytotoxicity under the same conditions (Figure S28). Together, these results suggest that D-BrushPEG achieves its antitumor efficacy through a dual contribution: partial backbone degradation appears to promote cellular uptake of the carrier, and the resulting degradation products could induce tumor cell death.

Safety and Clearance of D-BrushPEG and BrushPEG

Throughout the tumor treatment, no significant body-weight changes were observed, indicating good systemic tolerance (Figure J). Blood samples were collected from mice on day 7 posttreatment. Serum biochemistry analysis showed no significant changes in alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), or creatinine (Cre) levels in any polymer-treated group, indicating the absence of apparent hepatic or renal dysfunction (Figure A). Histopathological analysis showed no noticeable toxic effects in the hearts, livers, spleens, or lungs across any treatment group (Figure B).

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Safety evaluation of GEM-conjugated polymers. (A) Serum biochemical analysis (ALT, AST, BUN, Cre) of mice after antitumor treatment (n = 5 biologically independent samples per group). (B) Representative H&E-stained sections of major organs collected 7 days posttreatment. (C) Representative kidney sections at higher magnification and (D) renal injury scores for different treatment groups. (E) Representative skin sections and (F) cutaneous injury scores for different treatment groups. Data are shown as mean ± SD in (A) and as mean ± SEM in (D, F). Statistical significance was calculated by one-way ANOVA with Tukey’s multiple comparisons test. ns, not significant; *P < 0.05; **P < 0.01.

Mild renal changes were observed in drug-treated mice, including slight glomerular depletion and tubular epithelial edema, but histology scores showed no significant differences among treatments (Figure C and D). Skin pathology showed distinguishable treatment-dependent responses. BrushPEG-treated mice exhibited the greatest degree of cutaneous injury, characterized by focal hair follicle loss and occasional epidermal blistering, whereas D-BrushPEG caused noticeably milder changes (Figure E and F). These differences could be related to the distinct clearance profiles of the polymers, with prolonged retention of BrushPEG in the skin potentially contributing to increased local tissue exposure and cutaneous damage. D-BrushPEG and free GEM induced similar levels of skin irritation, suggesting that cutaneous damage was primarily driven by GEM rather than polymer structure. Overall, these findings indicate that the degradable architecture of D-BrushPEG helps reduce long-term tissue accumulation without increasing renal or systemic toxicity, although drug-related local side effects remain an important consideration independent of carrier design.

Given the potent antitumor activity of D-BrushPEG-GEM and the observed cytotoxicity of unconjugated D-BrushPEG in vitro, we next evaluated the safety of both brush polymers without GEM. A total polymer dose matching the total amount used in the tumor therapy study (1.2 μmol per mouse) was delivered to female Balb/c-nu mice through a regimen of three tail vein injections administered every other day (Figure A). Serum biochemistry and body weight analysis showed negligible differences compared to PBS treatment (Figure B–D). Histopathological evaluation of kidney tissues showed substantially lower pathological scores compared to the drug-loaded formulations, indicating that the previously observed nephrotoxic changes in the therapy study were predominantly associated with drug treatment rather than the polymer scaffolds themselves (Figure E and F). These findings suggest that the concentrations required to induce cytotoxicity are not reached in major organs following systemic administration.

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Safety evaluation and clearance of polymers (A) Treatment regimen for drug-free polymers in Balb/c-nu mice (n = 3 biologically independent samples per group). (B) Body-weight changes during treatment. (C) Serum biochemical analysis of liver-related (ALT, AST) and (D) renal-related (BUN, Cre) parameters. Dotted lines indicate corresponding values in untreated mice. (E) Representative H&E-stained kidney sections 7 days posttreatment. (F) Renal injury scores. (G) Representative IVIS images and (H) quantitative ROI analysis of in vivo fluorescence following subcutaneous injection of free Cy5, Cy5-labeled D-BrushPEG, or Cy5-labeled BrushPEG in C57BL/6 mice at various time points (10 nmol Cy5 per mouse, n = 4 biologically independent samples per group). Data are shown as mean ± SD in (B–D, H) and as mean ± SEM in (F).

Although BrushPEG did not induce acute toxicity, its nondegradable nature poses a potential risk of long-term bioaccumulation. To assess clearance, mice were subcutaneously injected with Cy5-labeled D-BrushPEG or BrushPEG, along with free Cy5 as a control. In vivo fluorescence imaging showed that D-BrushPEG was almost completely degraded and cleared within 10 days, whereas BrushPEG maintained strong fluorescence signals for more than a month with minimal decline after reaching its maximum (Figure G and 7H). These results highlight the clearance advantage of incorporating degradable motifs into bottlebrush architectures and underscore the potential risks associated with long-term persistence of nondegradable carriers.

Conclusion

This study introduces a hydrolytically degradable PEG bottlebrush polymer (D-BrushPEG) that addresses the long-standing challenge between circulation stability and clearance. By incorporating ODAN monomers into the bottlebrush backbone, D-BrushPEG maintains the prolonged systemic exposure characteristic of PEGylated brush architectures (elimination half-life ≈24.7 h), yet undergoes backbone hydrolysis that enables efficient elimination from the bloodstream. In contrast, a nondegradable BrushPEG analog displays a longer half-life and persistent retention. Beyond its pharmacokinetic advantages, D-BrushPEG exhibits enhanced cellular uptake and, when conjugated with gemcitabine via a reduction-responsive self-immolative linker, achieves notable antitumor efficacy in a HeLa xenograft model. Although no statistically significant difference in tumor inhibition was observed between BrushPEG and D-BrushPEG, D-BrushPEG maintained therapeutic efficacy while exhibiting improved clearance behavior. Safety evaluation indicates no overt hepatic or renal dysfunction and only mild, predominantly drug-associated renal and cutaneous changes, while the degradable architecture reduces polymer-related skin irritation compared with the nondegradable brush.

Collectively, these findings demonstrate that integrating hydrolytically labile units into PEG bottlebrush backbones can decouple long-circulating behavior from long-term tissue persistence, highlighting that backbone hydrolysis is a design parameter that can be used to shape pharmacokinetics, tumor delivery, and tissue exposure. The D-BrushPEG thus provides a platform for developing brush polymer therapeutics that more effectively balance delivery performance with clearance.

Experimental Methods

General Characterization

1H NMR, 13C NMR and 19F NMR spectra were acquired on a Bruker Fourier 300, Bruker Avance III 400 HD, or Bruker Avance 400 spectrometer in CDCl3 or DMSO-d 6. MestReNova software was used for data processing. High resolution mass spectrometry analyses were performed by ESI-MS (Bruker Solarix). Polymer solutions were prepared in tetrahydrofuran (THF) at a concentration of 1 mg/mL, filtered through a 0.45 μm nylon filter, and then subjected to GPC analysis. Number-average molecular weight (Mn) and polydispersity (PDI) were measured by GPC (Agilent 1260 Infinity II) in THF mobile phase at a flow rate of 0.3 mL/min. The molecular weight was calculated based on polystyrene standard. Hydrodynamic diameter and zeta potential were measured using a Zetasizer Nano ZS (Malvern) instrument equipped with a 632.8 nm laser by dissolving polymers in H2O.

Synthesis of Polymer Backbone

A solution of 47.0 mg of norbornene-NHS (0.2 mmol) in 200 μL of THF was prepared. For degradable polymers, 72.6 mg of ODAN (0.3 mmol) was added, followed by the addition of 1.45 mg of Grubbs III catalyst dissolved in 200 μL of THF. The reaction mixture was stirred at room temperature for 4 h. The reaction was quenched with vinyl ether, and 500 μL of DCM was added to fully re-dissolve the mixture. To remove metal ions, SiliaMetS was introduced, and stirring continued for 30 min at room temperature. The mixture was subsequently filtered and concentrated under reduced pressure, yielding the product in 98.1% yield.

Synthesis of Brush Polymer

The grafting reaction was performed by dissolving the backbone polymer (DPB or NPB) together with designated molar equivalents of PEG-amine, Cy5-amine or gemcitabine-linker-amine, and triethylamine (as a base catalyst) in anhydrous DMF. The reaction was allowed to proceed at 4 °C for 4 h under an inert atmosphere, with the consumption of free amine groups monitored qualitatively by ninhydrin hydrate staining. Upon completion, the product was isolated by precipitation into an excess of ice-cold diethyl ether. The mixture was centrifuged at 4,000g for 5 min, the supernatant was decanted, and the pellet was resuspended and washed with fresh cold ether. This wash cycle was repeated twice. The final precipitate was collected and dried under reduced pressure to yield the brush polymer as a white solid.

The details for the synthesis of other compounds are all represented in the Supporting Information.

Transmission Electron Microscopy (TEM) Analysis

A 5 μL aliquot of an aqueous solution of D-BrushPEG or BrushPEG (1 mg/mL) was deposited onto a 200-mesh copper grid pre-coated with a carbon support film. After an incubation period of 5 min, excess liquid was carefully blotted using filter paper. The grid was then stained with 5 μL of uranyl acetate solution (0.7 wt %), followed by gentle removal of residual stain. Subsequently, the grid was air-dried for a minimum of 5 min prior to imaging. TEM observations were carried out using an HT-7700 instrument (Hitachi, Japan) operated at an accelerating voltage of 100 kV.

Degradation of Bottlebrush Polymers under Buffered Conditions

To monitor the degradation profile of the polymer, 10 mg of the material was dissolved in 1 mL of PBS and incubated at 37 °C. Aliquots were collected at predetermined time intervals for DLS size analysis. For GPC characterization, the samples were first lyophilized. The resulting solid was re-dissolved in dichloromethane (DCM), dried over anhydrous sodium sulfate to remove residual water, and filtered through a 0.45 μm membrane. After solvent evaporation, the sample was re-dissolved in tetrahydrofuran (THF), filtered again through a 0.45 μm filter, and subsequently analyzed by GPC.

Quantification of Cy5-Labeled Polymer

A standard curve of Cyanine5 (Cy5) was established by measuring the fluorescence intensity (excitation: 650 nm, emission: 680 nm) of serially diluted Cy5 solutions in PBS (pH 7.4) using a BioTek Synergy H1 multimode microplate reader (Agilent). The Cy5-labeled polymer was dissolved in PBS and subsequently analyzed under the same instrumental settings. The concentration of Cy5-brush polymer was determined via interpolation against the established standard curve.

Cellular Uptake of Polymers

DC2.4 cells were maintained in RPMI 1640 medium containing 10% FBS and 1% p/s. The cells were seeded into 48-well plates at a density of 1 × 105 cells per well and cultured overnight at 37 °C under a humidified 5% CO2 atmosphere. Subsequently, the cells were incubated with Cy5-labeled polymers containing 20 ng Cy5 per well for 4, 24, or 48 h. Following treatment, the cells were harvested by trypsinization, washed with PBS, and analyzed by flow cytometry (CytoFLEX, Beckman Coulter).

Investigation of Cellular Internalization Pathways

To investigate the cellular internalization mechanisms, DC2.4 cells were pretreated with pathway-specific inhibitors for 30 min at 37 °C prior to incubation with Cy5-labeled polymers (containing 20 ng Cy5 per well) for 4 h under standard culture conditions. Following the treatment period, cells were detached using trypsinization, thoroughly washed with PBS, and subsequently analyzed by flow cytometry to quantify cellular uptake efficiency.

Animal Experiment

All animal experiments reported here were performed according to a protocol approved by the Committee of Biosafety, Ethics, and Laboratory Animal Management, IBP of CAS (Approval No. SYXK2025273).

In Vivo Circulation Half-Life of the Polymers

To assess the in vivo circulation kinetics of the polymer, ICR mice received a single intravenous injection of Cy5-labeled polymers (10 nmol Cy5 per mouse) via the tail vein. Blood samples were collected at predetermined time points using anticoagulant-treated tubes. Plasma was isolated by centrifugation, and the fluorescence intensity (excitation/emission: 650/680 nm) was measured using a microplate reader.

Establishment of a Subcutaneous HeLa Xenograft Tumor Model in Mice

HeLa cells (1 × 106) were subcutaneously inoculated into the right flank of 6–8-week-old Balb/c nu mice. Tumor growth was monitored every 3 days post-inoculation. Tumor volume was calculated using the formula: Volume = (Length × Width2)/2.

Tissue Distribution of Polymers in Tumor-Bearing Mice

Upon successful development of subcutaneous HeLa tumors reaching volumes of 50–80 mm3, the mice were administered a single intravenous injection of the Cy5-labeled polymers (10 nmol Cy5 per mouse). The mice were imaged by an in vivo imaging system (IVIS, PerkinElmer) at predetermined time points. At the experimental end point (13 days post-injection), terminal blood samples were collected, followed by humane euthanasia of the mice. Major organs (hearts, livers, spleens, lungs, and kidneys) together with tumor and skin tissues were then excised for ex vivo fluorescence imaging to assess biodistribution.

GEM Release from Polymer

A series of standard GEM solutions with gradient concentrations was prepared in PBS. GEM solutions were then subjected to High Performance Liquid Chromatography (HPLC) analysis on a Waters Breeze HPLC system equipped with a SunFire C18 column (5 μm, 4.6 × 150 mm) and a photodiode array detector. The mobile phase consisted of acetonitrile and triethylamine acetate buffer (0.1 M). A standard curve was established by plotting the concentration of GEM against the corresponding peak area. To evaluate the GEM release capability of GEM-loaded polymers under reductive conditions, the polymers were dissolved in PBS and dialyzed (using a 2 kDa dialysis membrane) against PBS with or without GSH (3 mM) at 37 °C. Samples were collected at predetermined time points and concentrated. The samples were subsequently analyzed by HPLC, and the concentrations were determined by interpolating the area under the corresponding peaks against the standard curve. The 100% release ratio was determined after co-incubating the samples with 30 mM GSH in PBS solution for 24 h.

In Vitro Cell Viability Assay

The in vitro cytotoxicity of the polymers was evaluated using HeLa cells. Cells were seeded in 96-well plates at a density of 5 × 103 cells per well in 100 μL of DMEM supplemented with 10% FBS and 1% p/s, and cultured overnight at 37 °C under a humidified 5% CO2 atmosphere. After the initial incubation, the cells were exposed to a range of concentrations of the polymers and incubated for 24 h. Subsequently, 10 μL of CCK-8 reagent was added to each well, followed by an additional 30 min incubation. Absorbance was measured at 450 nm with a reference wavelength of 690 nm using a microplate reader. Cell viability was calculated as the percentage of the absorbance in treated wells relative to untreated control wells (cells with culture medium only).

In Vivo Antitumor Efficacy Study

Upon the establishment of subcutaneous HeLa tumors reaching volumes of 50–80 mm3, mice were administered multiple intravenous doses of either GEM-loaded polymers or free GEM (5 mg/kg) at 3-day intervals. The animals were euthanized 7 days after the final administration, and blood along with major organs were collected for subsequent analysis.

Serum Biochemistry Analysis

Blood samples were collected and allowed to clot at room temperature for 2 h and subsequently centrifuged at 3,000g for 15 min to separate serum. Biochemical indicators, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine (Cre), and urea nitrogen (BUN), were measured by automatic biochemistry analyzer (Hitachi 7180). All procedures were performed in compliance with institutional guidelines for animal welfare.

H&E Staining

Major organs (hearts, livers, spleens, lungs, and kidneys) were harvested and then fixed in 4% paraformaldehyde and embedded in paraffin. Paraffin-embedded tissues were sliced into 5 μm sections and stained by hematoxylin and eosin (H&E). The slides were scanned by the digital slide scanner (HAMAMATSU Nanozoomer 2.0-RS). The grade of renal injury was assessed using a seven-tiered grading system, evaluated based on the degree of renal tubular epithelial cell necrosis, loss of the tubular brush border, and reduction in glomerular number. Similarly, the extent of skin injury was classified into seven grades according to the severity of hair follicle loss in the subcutaneous tissue and the degree of subepidermal blister formation.

Evaluation of Drug-Free Polymers Biocompatibility

6–8-week-old female Balb/c-nu mice were administered the drug-free polymers via intravenous injection at a dose of 400 nmol per injection every other day, which is approximately twice the dose per injection used in the tumor therapy experiments. Tissues were collected on day 7 following the final administration for subsequent analysis.

Long-Term Monitoring of Polymers Metabolism

To evaluate the long-term pharmacokinetics behavior of the polymers, C57BL/6 mice were subcutaneously administered Cy5-labeled polymers (10 nmol Cy5 per mouse). Non-invasive in vivo imaging was performed at predetermined time points using an IVIS imaging system.

Supplementary Material

ps6c00029_si_001.pdf (1.9MB, pdf)

Acknowledgments

This research was supported by the National Key Research and Development Program of China (No. 2024YFA1212200), the Strategic Priority Research Program of the Chinese Academy of Sciences (No. XDB0960100), the International Partnership Program of the Chinese Academy of Sciences for Grand Challenges (No. 027GJHZ2023179GC), and the National Natural Science Foundation of China (No. 22175188, 22575254) to X.L.

The data that support the findings of this study are available from the corresponding author upon reasonable request.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/polymscitech.6c00029.

  • Materials, synthetic procedures, NMR and mass spectra, GEM released under oxidative conditions, and cytotoxicity data of D-BrushPEG and BrushPEG (PDF)

The authors declare no competing financial interest.

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Associated Data

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

Supplementary Materials

ps6c00029_si_001.pdf (1.9MB, pdf)

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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