Postpolymerization Modification of Poly(2-vinyl-4,4-dimethyl azlactone) as a Versatile Strategy for Drug Conjugation and Stimuli-Responsive Release

Abstract

Postpolymerization modification of highly defined "scaffold" polymers is a promising approach for overcoming the existing limitations of controlled radical polymerization such as batch-to-batch inconsistencies, accessibility to different monomers, and compatibility with harsh synthesis conditions. Using multiple physicochemical characterization techniques, we demonstrate that poly(2-vinyl-4,4-dimethyl azlactone) (PVDMA) scaffolds can be efficiently modified with a coumarin derivative, doxorubicin, and camptothecin small molecule drugs. Subsequently, we show that coumarin-modified PVDMA has a high cellular biocompatibility and that coumarin derivatives are liberated from the polymer in the intracellular environment for cytosolic accumulation. In addition, we report the pharmacokinetics, biodistribution, and antitumor efficacy of a PVDMA-based polymer for the first time, demonstrating unique accumulation patterns based on the administration route (i.e., intravenous vs oral), efficient tumor uptake, and tumor growth inhibition in 4T1 orthotopic triple negative breast cancer (TNBC) xenografts. This work establishes the utility of PVDMA as a versatile chemical platform for producing polymer-drug conjugates with a tunable, stimuli-responsive delivery.

Figure 1.. Post-polymerization modification of PVDMA with DBAC and doxorubicin.

a) ¹H-NMR spectra of DBAC-modified PVDMA polymer in MeOD, (*) solvent peak, b) FTIR spectrum of homopolymer PVDMA, DBAC-modified PVDMA, c) ¹H-NMR spectra of doxorubicin-modified PVDMA polymer in DMSO-d6, (*) solvent peak, d) FTIR spectrum of homopolymer PVDMA, free doxorubicin, and doxorubicin-modified PVDMA polymer, e) Schematic of DBAC hydrolysis and release from DBAC-modified PVDMA, f) In-vitro release profile of DBAC from DBAC-PVDMA polymers at pH values of 5.5 and 7.4. Data are expressed as mean ± SD (n =3) and analyzed by Student’s t-test: Bars with different letters indicate significant (P< 0.05) difference between the pH’s.

Figure 2.. Biocompatibility and cell uptake of DBAC-PVDMA polymers.

(a) Cell viability of HeLa cells after incubation with varying concentrations of DBAC-PVDMA (P3) for 24 h (Data represent mean ± s.d., n=4), (b) Cell viability of HEK-293 cells after incubation with different concentrations of DBAC-PVDMA for 24 h (Data represent mean ± s.d., n=4), c) Schematic synthesis diagram of fluorescently-labeled PVDMA polymer (DBAC-TMR-PVDMA), confocal microscopy images of DBAC-TMR-PVDMA in HEK-293 cells d) upon 10 min treatment, e) upon overnight incubation, and f) time-lapse following 10 min treatment.

Figure 3.. Subcellular colocalization of DBAC and TMR-PVDMA polymer.

(a) Fluorescence imaging showing the distribution of DBAC (C), TMR (R), and LysoTracker Green (L) dyes in HEK-293 cells. (b) Fluorescence imaging showing the overlap of all three dyes in HEK-293 cells. DBAC, TMR, and LysoTracker Green dyes, (c) Fluorescence imaging showing the overlap between 2 dyes in HEK-293 cells. Overlap of DBAC and TMR (CR), DBAC and LysoTracker Green (CL), and TMR and LysoTracker Green (RL) dyes, respectively. (d) Pearson’s correlation graph showing the colocalization relationship between two dyes (left-to-right: CR, CL, and RL). ANOVA followed by Tukey’s test where different letters indicate statistical significance (P< 0.05).

Figure 4.. IV Injection of DBAC-TMR-PVDMA extends DBAC circulation and drives kidney accumulation.

Pharmacokinetics of DBAC-TMR-PVDMA polymers in (a) plasma, (b) lungs, (c) heart, (d) spleen, (e) liver, and (f) kidney after i.v. injection. (Data represent mean ± s.d., n=3 at each time point)

Figure 5.. Oral administration of DBAC-TMR-PVDMA drives high accumulation in the GI tract with limited systemic absorption.

Pharmacokinetics of DBAC-TMR-PVDMA polymers in the (a) GI tract, (b) plasma, (c) lungs, (d) kidney, (e) liver, and (f) spleen after oral administration. (Data represent mean ± s.d., n=3 at each time point)

Figure 6.. Acute biocompatibility analysis of DBAC-PVDMA after intravenous administration.

(a) Body mass of Balb/C mice over time following intravenous injection of saline or DBAC-PVDMA at 10 and 33.3 mg/kg (n = 4). Serum levels of (b) BUN (c) AST (d) ALT, and (e) ALP following intravenous injection of saline or DBAC-PVDMA at 10 and 33.3 mg/kg (n = 4). (f) Representative histopathology images of major organs (liver, heart, and kidneys) following intravenous injection of saline or DBAC-PVDMA (Scale Bar = 200 μm). Data are expressed as mean ± SD (n =4) and analyzed by one-way ANOVA followed by Tukey’s test: Bars with different letters indicate significant (P< 0.05) difference between the group. Changes in the treatment are represented with respect to the control.

Figure 6.. Acute biocompatibility analysis of DBAC-PVDMA after intravenous administration.

(a) Body mass of Balb/C mice over time following intravenous injection of saline or DBAC-PVDMA at 10 and 33.3 mg/kg (n = 4). Serum levels of (b) BUN (c) AST (d) ALT, and (e) ALP following intravenous injection of saline or DBAC-PVDMA at 10 and 33.3 mg/kg (n = 4). (f) Representative histopathology images of major organs (liver, heart, and kidneys) following intravenous injection of saline or DBAC-PVDMA (Scale Bar = 200 μm). Data are expressed as mean ± SD (n =4) and analyzed by one-way ANOVA followed by Tukey’s test: Bars with different letters indicate significant (P< 0.05) difference between the group. Changes in the treatment are represented with respect to the control.

Figure 7.. DBAC-PVDMA conjugates exhibit efficient tumor accumulation and anti-tumor efficacy in TNBC xenografts.

(a) Quantification of DBAC-TMR-PVDMA levels in major organs and tumor 24 hours following intravenous administration at 10 mg/kg dose (n = 4). (b) Representative IVIS image of DBAC-TMR-PVDMA accumulation in major organs and tumor. (c) Treatment protocol for anti-tumor efficacy study in 4T1 orthotopic mammary xenografts. (d) Tumor growth curves for saline, DBAC-PVDMA (10 mg/kg), and DBAC (equivalent dose) treatment of 4T1 tumor-bearing mice (n = 4). (e) Image of tumor sizes at study endpoint (n = 4). Data are expressed as mean ± SD (n =4) and analyzed by one-way ANOVA followed by Tukey’s test: Bars with different letters indicate significant (P< 0.05) difference between the group. Changes in the treatment are represented with respect to the control.

Scheme 1:

Synthetic pathways for a) the PVDMA polymer scaffold, b) Post-polymerization modification of the PVDMA scaffold by DBAC, c) Post-polymerization modification of PVDMA by doxorubicin, and d) Post-polymerization modification of PVDMA by camptothecin.