pH-Responsive Poly(ethylene glycol)-b-poly(2-vinylpyridine) Micelles for the Triggered Release of Therapeutics

The use of pH-responsive polymeric micelles is a promising approach to afford the targeted, pH-mediated delivery of hydrophobic drugs within the low-pH tumour milieu and intracellular organelles of cancer cells. However, even for a common pH-responsive polymeric micelle system—e.g., those utilising poly(ethylene glycol)-b-poly(2-vinylpyridine) (PEG-b-PVP) diblock copolymers—there is a lack of available data describing the compatibility of hydrophobic drugs, as well as the relationships between copolymer microstructure and drug compatibility. Furthermore, synthesis of the constituent pH-responsive copolymers generally requires complex temperature control or degassing procedures that limit their accessibility. Herein we report the facile synthesis of a series of diblock copolymers via visible-light-mediated photocontrolled reversible addition-fragmentation chain-transfer polymerisation, with a constant PEG block length (90 repeat units (RUs)) and varying PVP block lengths (46–235 RUs). All copolymers exhibited narrow dispersity values (Đ ≤ 1.23) and formed polymeric micelles with low polydispersity index (PDI) values (typically <0.20) at physiological pH (7.4), within a suitable size range for passive tumour targeting (<130 nm). The encapsulation and release of three hydrophobic drugs (cyclin-dependent kinase inhibitor (CDKI)-73, gossypol, and doxorubicin) were investigated in vitro at pH 7.4–4.5 to simulate drug release within the tumour milieu and cancer cell endosome. Marked differences in drug encapsulation and release were observed when the PVP block length was increased from 86 to 235 RUs. With a PVP block length of 235 RUs, the micelles exhibited differing encapsulation and release properties for each drug. Minimal release was observed for doxorubicin (10%, pH 4.5) and CDKI-73 exhibited moderate release (77%, pH 4.5), whereas gossypol exhibited the best combination of encapsulation efficiency (83%) and release (91% pH 4.5) overall. These data demonstrate the drug selectivity of the PVP core, where both the block molecular weight and hydrophobicity of the core (and accordingly the hydrophobicity of the drug) have a significant effect on drug encapsulation and release. These systems remain a promising means of achieving targeted, pH-responsive drug delivery—albeit for select, compatible hydrophobic drugs—which warrants their further investigation to develop and evaluate clinically relevant micelle systems.


Introduction
The effective delivery of poorly bioavailable, hydrophobic drugs for anti-cancer treatments is a persistent challenge within the pharmaceutical sector. pH-responsive polymeric micelles have shown considerable promise as a means of achieving targeted and controlled delivery [1,2]. For site-specific drug release, block copolymers and their corresponding micelles can be engineered to exploit the pH differential existing between healthy tissues (pH 7.4) and the tumour milieu (pH~6. 5-7.2) or the intracellular organelles of cancerous cells (endo/lysosome pH 5.0-6.5) to trigger drug release [1,3,4]. Cancerous tumours can constitutive pH-responsive copolymers. This work demonstrates the applicability of the photo-RAFT approach for the downstream preparation of micelles and provides valuable insight into the compatibility of hydrophobic drugs with PEG-b-PVP micelles, and the structural features governing this compatibility.
PBS (pH 7.4 and 6.5) solutions were prepared following the manufacturer's guidelines, with final pH adjustments made via the addition of 0.1 M HCl. Deuterated PBS was prepared using a PBS tablet and D 2 O, following the manufacturer's instructions for the preparation of non-deuterated PBS. Buffered solutions (pH 5.5 and 4.5) were prepared using a citric acid/sodium citrate buffer, prepared according to the United States Pharmacopeia (USP 29).

Characterisation
The composition and number-average molecular weights (M n ) of the PEG x PVP y copolymers were determined via 1 H NMR spectroscopy using a Bruker Avance III HD 500 NMR spectrometer (Billerica, MA, USA), operating at 500 MHz, and calculated using the ratios of the integral values of the characteristic resonances at δ H 3.63 (PEG RU), 3.36 (PEG methoxy end-group), and 8.20 (PVP RU) ppm.
The molecular weight characteristics of the polymers, including M n , weight-average molecular weight (M w ) and Ð were determined via gel permeation chromatography (GPC), using a Shimadzu Prominence liquid chromatography system (Kyoto, Japan) fitted with a refractive index detector (Shimadzu, RID-20A), two mixed gel columns in series (Shimadzu GPC-80MD and GPC-804D), and using THF as the mobile phase (1 mL·min −1 , 40 • C). A conventional column calibration was performed with a series of narrow molecular weight polystyrene standards (Polymer Standards Service GmbH, Mainz, Germany).
Copolymer syntheses were monitored, and monomer conversion was determined via gas chromatography (Shimadzu GC-2010 fitted with a flame ionisation detector (FID)), based on pre-polymerisation concentrations, using the solvent (DMSO or MeOH) as an internal reference. Aliquots of the crude reaction mixture were diluted in DCM (~1.5 mL) prior to analysis and introduced using a split injection (50:1 ratio) to a Supelco SPB-35 stationary phase column (30.0 m × 0.25 mm, 0.25 µm). The GC was operated in linear velocity mode with nitrogen (BOC, 99.999%) as the carrier gas (56.5 cm·s −1 ), with injection port and FID temperatures of 250 • C, and a temperature program of 35 • C (1 min hold) to 185 • C (1 min hold) at 30.0 • C·min −1 .
Particle size distributions were measured via dynamic light scattering using a Malvern Zetasizer Nano ZS (He-Ne laser, λ = 633 nm, 4 mW) (Malvern, United Kingdom), using a cell temperature of 25 • C.
Drug concentrations from release experiments were quantified via UV-visible spectrophotometry using a Thermo Fisher Evolution 260 Bio (Waltham, MA, USA), fitted with a Thermo Fisher Peltier Control Cooling Unit, and operating at a cell temperature of 25 • C). Absorbances were determined at the lambda maximum for each drug (CDKI-73 = 275 nm, doxorubicin = 494 nm, and gossypol = 386 nm). Standard curves were generated across the concentration range of 0.5 to 12 µg·mL −1 , and drug concentrations were quantified using molar absorption coefficients (ε) determined in each buffered solution (pH 7.4-4.5), for each drug (supporting information (SI), Table S1).
The pH values of the solutions were measured throughout using an Oakton pH 700 pH meter (Environmental Express, Charleston, SC, USA) fitted with an Oakton All-in-One pH/ATC Probe.

Polymeric Micelle Preparation
Polymeric micelles were prepared from the PEG x PVP y copolymers via the co-solvent evaporation approach. An equal volume of PBS (10 mM, pH 7.4) was added dropwise to the copolymer solution (1 mg·mL −1 , acetone) under sonication. The mixture was then heated at 60 • C until the acetone had evaporated to afford a 1 mg·mL −1 micellar solution. The resultant solutions were pH-adjusted via the addition of small volumes of 0.1 M HCl to afford micellar solutions of~1 mg·mL −1 at pH 4.0-7.4.

1 H NMR Spectroscopy of Polymeric Micelles
Polymeric micelles were prepared from PEG 90 PVP 235 as described above at pH 7.

Drug Encapsulation and Release
Copolymers (PEG 90 PVP 235 or PEG 90 PVP 86 ) were solubilised in acetone with the target drug (CDKI-73, doxorubicin, or gossypol) to afford concentrations of 0.9 and 0.1 mg·mL −1 , respectively. Polymeric micelles were then prepared as previously described to afford 1 mg·mL −1 drug-loaded micellar solutions in PBS (10 mM, pH 7.4) with a drug loading of 10 wt%. Drug-loaded micellar solutions (4 mL) were then placed in dialysis tubing (7 kDa MWCO) and suspended in a pH 7.4 receiving solution (40 mL, 15 h) to remove any unencapsulated drug. The receiving solution was then replaced with buffered solutions of decreasing pH every 3 h (pH 6.5, 5.5, and 4.5) to investigate the release of the encapsulated drug. Aliquots of the receiving solution (1 mL) were removed hourly and analysed using UV-visible spectrophotometry to quantify drug release. The total volume of the receiving solution was maintained throughout the experiment via the addition of 1 mL of buffered solution at the appropriate pH.

Results and Discussion
Using a trithiocarbonate RAFT macroinitiator (PEG-DDMAT) (Supplementary Materials, Figure S2), a series of linear diblock PEG-b-PVP copolymers were synthesised via visible-light-mediated RAFT (photo-RAFT) polymerisation [26] using a 460 nm visible light source (Scheme 1). The polymerisation was stopped after defined periods by switching off the light source, allowing for control over the monomer conversion and the PVP block length ( Table 1). As reported previously by Fu et al. for the photo-RAFT of acrylates, the inclusion of a sacrificial amine (triethanolamine) in combination with trithiocarbonate as a photoredox catalyst for deoxygenation and polymerisation, enabled the well-controlled, oxygen-tolerant photo-RAFT of vinylarenes (i.e., vinylpyridine) from trithiocarbonate macroinitiators.
controlled, oxygen-tolerant photo-RAFT of vinylarenes (i.e., vinylpyridine) from trithiocarbonate macroinitiators. Scheme 1. Preparation of PEG-b-PVP copolymers via the photo-RAFT of 2-vinylpyridine from the PEG-DDMAT macroinitiator, and the pH-mediated formation and disassembly of their drug-loaded micelles. A series of copolymers were prepared with a common PEG-DDMAT initiator (90 RU, Mn = 4.3 kDa) to allow the effect of the PVP block length on micelle hydrodynamic diameter (Dh), pH-responsiveness, and drug encapsulation and release to be investigated. PEGb-PVP copolymers were synthesised with PVP block lengths and weight fractions of PVP (fPVP) ranging from 46-235 RU and 0.53-0.85, respectively ( Table 1). The copolymers were referred to as PEGxPVPy, where x and y refers to the number of PEG and PVP RUs, respectively. 1 Figure S1), indicating good control over the polymerisation.
Micelles were prepared in PBS (10 mM, pH 7.4, 1 mg·mL −1 ) from the copolymers using the co-solvent evaporation approach, then pH-adjusted to afford solutions with pH values ranging from 7.4 to 4.0. Particle size distributions (PSD) determined by DLS revealed the successful self-assembly of copolymers into micelles (Figure 1), which were stable for at least several days following preparation and storage at 23 °C. At physiological pH (7.4), the number-weighted distributions showed that the Dh values of the micelles increased with increasing fPVP of the copolymers, which ranged from 22 nm (PEG90PVP46) Scheme 1. Preparation of PEG-b-PVP copolymers via the photo-RAFT of 2-vinylpyridine from the PEG-DDMAT macroinitiator, and the pH-mediated formation and disassembly of their drug-loaded micelles. A series of copolymers were prepared with a common PEG-DDMAT initiator (90 RU, M n = 4.3 kDa) to allow the effect of the PVP block length on micelle hydrodynamic diameter (D h ), pH-responsiveness, and drug encapsulation and release to be investigated. PEG-b-PVP copolymers were synthesised with PVP block lengths and weight fractions of PVP (f PVP ) ranging from 46-235 RU and 0.53-0.85, respectively ( Table 1). The copolymers were referred to as PEG x PVP y , where x and y refers to the number of PEG and PVP RUs, respectively. 1 H NMR spectroscopic analysis of the copolymers (Supplementary Materials, Figure S5) provided M n values consistent with the theoretical M n values determined from GC quantification of the monomer conversions. GPC of the copolymers provided Ð values of ≤1.23 for all copolymers (Supplementary Materials, Figure S1), indicating good control over the polymerisation.
Micelles were prepared in PBS (10 mM, pH 7.4, 1 mg·mL −1 ) from the copolymers using the co-solvent evaporation approach, then pH-adjusted to afford solutions with pH values ranging from 7.4 to 4.0. Particle size distributions (PSD) determined by DLS revealed the successful self-assembly of copolymers into micelles (Figure 1), which were stable for at least several days following preparation and storage at 23 • C. At physiological pH (7.4), the number-weighted distributions showed that the D h values of the micelles increased with increasing f PVP of the copolymers, which ranged from 22 Table S3).  Figure S6 and Table S2). Values are reported as mean + std. dev.
The fPVP of the copolymers were positively correlated with both the Dh and PDI of the micelle populations at pH 7.4 ( Figure 1 and Supplementary Materials, Tables S2 and S3). These trends are consistent with previous studies, whereby smaller, well-defined micelle morphologies were reported for PEG-b-PVP copolymers (prepared from both VP and 4vinylpyridine (4VP)) in which the PEG block predominated, and comparatively poorly defined and larger micellar aggregates or vesicles formed when the PVP block predominated [10,12].
When the pH of the micellar solutions was decreased, a gradual decline in Dh was observed at pH 6.0-5.5, which correlates to protonation/ionisation of the PVP block as the pH approaches the pKa of VP (pKa ~ 5) and previously reported pKa values for PEG-b-PVP copolymers [12,13,17]. Protonation of the PVP block would be expected to cause micelle swelling, reorganisation, and disassembly, until unimers are observed at a sufficiently low pH. For the higher fPVP copolymers (i.e., PEG90PVP152 and PEG90PVP235), disassembly was preceded by a slight increase in Dh at pH 6.0. This is likely due to the presence of partially protonated, and electrostatically repulsive PVP blocks at the perimeter of the micelle core, hindering the steric stabilisation afforded by the PEG blocks and causing swelling and an  Figure S6 and Table S2). Values are reported as mean + std. dev.
The f PVP of the copolymers were positively correlated with both the D h and PDI of the micelle populations at pH 7.4 ( Figure 1 and Supplementary Materials, Tables S2 and S3). These trends are consistent with previous studies, whereby smaller, well-defined micelle morphologies were reported for PEG-b-PVP copolymers (prepared from both VP and 4-vinylpyridine (4VP)) in which the PEG block predominated, and comparatively poorly defined and larger micellar aggregates or vesicles formed when the PVP block predominated [10,12].
When the pH of the micellar solutions was decreased, a gradual decline in D h was observed at pH 6.0-5.5, which correlates to protonation/ionisation of the PVP block as the pH approaches the pK a of VP (pK a~5 ) and previously reported pK a values for PEG-b-PVP copolymers [12,13,17]. Protonation of the PVP block would be expected to cause micelle swelling, reorganisation, and disassembly, until unimers are observed at a sufficiently low pH. For the higher f PVP copolymers (i.e., PEG 90 PVP 152 and PEG 90 PVP 235 ), disassembly was preceded by a slight increase in D h at pH 6.0. This is likely due to the presence of partially protonated, and electrostatically repulsive PVP blocks at the perimeter of the micelle core, hindering the steric stabilisation afforded by the PEG blocks and causing swelling and an increase in micelle size. This observation is consistent with previously reported results for micelles containing PVP [7,12,13,17]. The same trend is not observed for copolymers with f PVP < 0.68 (i.e., PEG 90 PVP 46 , PEG 90 PVP 58 , and PEG 90 PVP 86 ), ostensibly due to the greater stability conferred by PEG in these micelles where a larger weight fraction of PEG exists. Regardless, further reduction in pH (and protonation of the PVP blocks) afforded an apparent disassembly of the micelles into smaller aggregates or unimers for all the studied copolymers (Table S2).
The DLS results of the micelle solutions were supported by 1 H NMR spectroscopy, which revealed protonation of the PVP block at low pH. For example, the NMR spectrum of PEG 90 PVP 235 in deuterated PBS (pH* 7.44, 1 mg·mL −1 ) revealed a resonance from the PEG block but no aromatic proton resonances (~8.5-6.5 ppm) from the neutral PVP block due to their poor solvation and restricted mobility within the tightly packed micelle core ( Figure 2). However, when the pH* was adjusted to 4.53, the PVP proton resonances were visible, due to their protonation and the disruption of the micelle structure (caused by electrostatic repulsion between protonated pyridinyl moieties), resulting in increased solvation and greater mobility. These observations were consistent with those reported by Atanase et al. [17] and indicated the successful assembly and disassembly of the micelles through protonation of the PVP block. increase in micelle size. This observation is consistent with previously reported results for micelles containing PVP [7,12,13,17]. The same trend is not observed for copolymers with fPVP < 0.68 (i.e., PEG90PVP46, PEG90PVP58, and PEG90PVP86), ostensibly due to the greater stability conferred by PEG in these micelles where a larger weight fraction of PEG exists. Regardless, further reduction in pH (and protonation of the PVP blocks) afforded an apparent disassembly of the micelles into smaller aggregates or unimers for all the studied copolymers (Table S2). The DLS results of the micelle solutions were supported by 1 H NMR spectroscopy, which revealed protonation of the PVP block at low pH. For example, the NMR spectrum of PEG90PVP235 in deuterated PBS (pH* 7.44, 1 mg·mL −1 ) revealed a resonance from the PEG block but no aromatic proton resonances (~ 8.5-6.5 ppm) from the neutral PVP block due to their poor solvation and restricted mobility within the tightly packed micelle core ( Figure 2). However, when the pH* was adjusted to 4.53, the PVP proton resonances were visible, due to their protonation and the disruption of the micelle structure (caused by electrostatic repulsion between protonated pyridinyl moieties), resulting in increased solvation and greater mobility. These observations were consistent with those reported by Atanase et al. [17] and indicated the successful assembly and disassembly of the micelles through protonation of the PVP block. The observed reorganisation and disassembly of the micelles between pH 6.0-5.5 implied that the micelles would be responsive to the endosomal environment [4], which could be manipulated as a trigger for the release of an encapsulated drug. To investigate this prospect and whether the physiochemical properties of therapeutics play a role in their triggered release from the micelles, we assessed the encapsulation and release of three hydrophobic drugs (Table 2) with the potential for cancer treatment, as a function of pH and fPVP. The observed reorganisation and disassembly of the micelles between pH 6.0-5.5 implied that the micelles would be responsive to the endosomal environment [4], which could be manipulated as a trigger for the release of an encapsulated drug. To investigate this prospect and whether the physiochemical properties of therapeutics play a role in their triggered release from the micelles, we assessed the encapsulation and release of three hydrophobic drugs (Table 2) with the potential for cancer treatment, as a function of pH and f PVP .  Drug-loaded polymeric micelles (10 wt% drug loading) were initially prepared using the PEG90PVP235 copolymer in PBS at pH 7.4 and dialysed against a pH 7.4 receiving solution for 15 h to assess for the presence of any unencapsulated drug. The pH of the receiving solution was then decreased by ~ 1 pH unit, every 3 h, to pH 4.5, to simulate the low pH environments of the cancerous tumour milieu and intracellular organelles (i.e., the endo/lysosome) [33].
All three drugs were successfully encapsulated in the PEG90PVP235 micelles; however, a marked difference in encapsulation and release was observed (Figure 3). Gossypol showed an idealised release profile, with 83% drug encapsulation efficiency (17% unencapsulated drug release at pH 7.4) and rapid release starting at pH 5.5 and reaching a maximum of 91% at pH 4.5, consistent with the pH-mediated disassembly of the polymeric micelles. In contrast, doxorubicin exhibited 100% encapsulation (no unencapsulated drug detected at pH 7.4) but failed to release a significant amount of drug (< 10% at pH 4.5). This difference was attributed to the hydrophobicity of both drugs and their noncovalent interactions with the PVP block. As gossypol is more hydrophobic compared to doxorubicin (Table 2), it is conceivable that the van der Waals forces present between gossypol and the micelle core are disrupted through protonation of the PVP blocks at low pH (≤ 5.5). Whereas, at higher pH values (≥ 6.5), van der Waals forces and H-bonding between the phenolic groups of gossypol and the PVP core would contribute to its retention. The increase in core hydrophilicity upon protonation of the PVP blocks likely results in  Drug-loaded polymeric micelles (10 wt% drug loading) were initially prepared using the PEG90PVP235 copolymer in PBS at pH 7.4 and dialysed against a pH 7.4 receiving solution for 15 h to assess for the presence of any unencapsulated drug. The pH of the receiving solution was then decreased by ~ 1 pH unit, every 3 h, to pH 4.5, to simulate the low pH environments of the cancerous tumour milieu and intracellular organelles (i.e., the endo/lysosome) [33].
All three drugs were successfully encapsulated in the PEG90PVP235 micelles; however, a marked difference in encapsulation and release was observed (Figure 3). Gossypol showed an idealised release profile, with 83% drug encapsulation efficiency (17% unencapsulated drug release at pH 7.4) and rapid release starting at pH 5.5 and reaching a maximum of 91% at pH 4.5, consistent with the pH-mediated disassembly of the polymeric micelles. In contrast, doxorubicin exhibited 100% encapsulation (no unencapsulated drug detected at pH 7.4) but failed to release a significant amount of drug (< 10% at pH 4.5). This difference was attributed to the hydrophobicity of both drugs and their noncovalent interactions with the PVP block. As gossypol is more hydrophobic compared to doxorubicin (Table 2), it is conceivable that the van der Waals forces present between gossypol and the micelle core are disrupted through protonation of the PVP blocks at low pH (≤ 5.5). Whereas, at higher pH values (≥ 6.5), van der Waals forces and H-bonding between the phenolic groups of gossypol and the PVP core would contribute to its retention. The increase in core hydrophilicity upon protonation of the PVP blocks likely results in  Drug-loaded polymeric micelles (10 wt% drug loading) were initially prepared using the PEG90PVP235 copolymer in PBS at pH 7.4 and dialysed against a pH 7.4 receiving solution for 15 h to assess for the presence of any unencapsulated drug. The pH of the receiving solution was then decreased by ~ 1 pH unit, every 3 h, to pH 4.5, to simulate the low pH environments of the cancerous tumour milieu and intracellular organelles (i.e., the endo/lysosome) [33].
All three drugs were successfully encapsulated in the PEG90PVP235 micelles; however, a marked difference in encapsulation and release was observed (Figure 3). Gossypol showed an idealised release profile, with 83% drug encapsulation efficiency (17% unencapsulated drug release at pH 7.4) and rapid release starting at pH 5.5 and reaching a maximum of 91% at pH 4.5, consistent with the pH-mediated disassembly of the polymeric micelles. In contrast, doxorubicin exhibited 100% encapsulation (no unencapsulated drug detected at pH 7.4) but failed to release a significant amount of drug (< 10% at pH 4.5). This difference was attributed to the hydrophobicity of both drugs and their noncovalent interactions with the PVP block. As gossypol is more hydrophobic compared to doxorubicin (Table 2), it is conceivable that the van der Waals forces present between gossypol and the micelle core are disrupted through protonation of the PVP blocks at low pH (≤ 5.5). Whereas, at higher pH values (≥ 6.5), van der Waals forces and H-bonding between the phenolic groups of gossypol and the PVP core would contribute to its retention. The increase in core hydrophilicity upon protonation of the PVP blocks likely results in Drug-loaded polymeric micelles (10 wt% drug loading) were initially prepared using the PEG 90 PVP 235 copolymer in PBS at pH 7.4 and dialysed against a pH 7.4 receiving solution for 15 h to assess for the presence of any unencapsulated drug. The pH of the receiving solution was then decreased by~1 pH unit, every 3 h, to pH 4.5, to simulate the low pH environments of the cancerous tumour milieu and intracellular organelles (i.e., the endo/lysosome) [33].
All three drugs were successfully encapsulated in the PEG 90 PVP 235 micelles; however, a marked difference in encapsulation and release was observed (Figure 3). Gossypol showed an idealised release profile, with 83% drug encapsulation efficiency (17% unencapsulated drug release at pH 7.4) and rapid release starting at pH 5.5 and reaching a maximum of 91% at pH 4.5, consistent with the pH-mediated disassembly of the polymeric micelles. In contrast, doxorubicin exhibited 100% encapsulation (no unencapsulated drug detected at pH 7.4) but failed to release a significant amount of drug (<10% at pH 4.5). This difference was attributed to the hydrophobicity of both drugs and their non-covalent interactions with the PVP block. As gossypol is more hydrophobic compared to doxorubicin (Table 2), it is conceivable that the van der Waals forces present between gossypol and the micelle core are disrupted through protonation of the PVP blocks at low pH (≤5.5). Whereas, at higher pH values (≥6.5), van der Waals forces and H-bonding between the phenolic groups of gossypol and the PVP core would contribute to its retention. The increase in core hydrophilicity upon protonation of the PVP blocks likely results in disruption of these non-covalent interactions causing micelle disassembly and the release of gossypol into the receiving solution.
Pharmaceutics 2023, 15, x FOR PEER REVIEW 10 of 13 disruption of these non-covalent interactions causing micelle disassembly and the release of gossypol into the receiving solution. Contrastingly, the more polar doxorubicin was likely retained due to the presence of π-π stacking with the PVP blocks, which could be attributed to the planar anthraquinone motif of doxorubicin (cf., gossypol), which is also responsible for the intercalation of doxorubicin with DNA [34,35]. At all tested solution pH values, the amine group of doxorubicin would be protonated. The electrostatic repulsion afforded at low pH between the protonated doxorubicin and PVP blocks was likely insufficient to induce complete micelle disassembly due to π-π stacking. The small amount of release observed for PEG90PVP235 micelles containing doxorubicin may be due to the loss of loosely bound doxorubicin, whilst the majority was retained.
The encapsulation and release of CDKI-73 exhibited a comparatively intermediate profile, with 100% encapsulation efficiency (no release detected at pH 7.4) and an approximately linear release profile with decreasing pH, reaching an overall release of 46%. However, a lack of available data makes it challenging to relate these results to the physicochemical properties of CDKI-73. An estimated LogP value of 2.92 for CDKI-73 (Table 2) suggests that these results are consistent with its intermediate hydrophobicity (cf. doxorubicin and gossypol). Of the various functional groups in CDKI-73, only the 5-aryl-2aminothiazole motif might be expected to have a pKa value that is of significance in this study. If it is assumed that CDKI-73 has a similar pKa value to the structural analogue, 5phenyl-2-aminothiazole (pKa = 4.9) [36], then CDKI-73 would become protonated and positively charged in parallel with PVP protonation/ionisation. This ionisation would cause electrostatic repulsion between CDKI-73 and the PVP blocks, driving micelle disassembly and drug release in the absence of other sufficiently strong non-covalent interactions (cf. doxorubicin). The observed trends for the three drugs indicated that very hydrophobic drugs are released rapidly upon a decrease in pH due to repulsion from the hydrophilic, protonated PVP blocks, whereas more polar drugs have a stronger attraction to the protonated PVP blocks which can impede release.
To assess the effect of the fPVP on encapsulation and release, CDKI-73-loaded micelles were prepared from PEG90PVP86 exhibiting a shorter PVP block length. A significant decrease in encapsulation efficiency (66%) was observed for the PEG90PVP86 micelles (cf. PEG90PVP235 micelles) (Figure 3), most likely because of the smaller fPVP not being sufficient Contrastingly, the more polar doxorubicin was likely retained due to the presence of π-π stacking with the PVP blocks, which could be attributed to the planar anthraquinone motif of doxorubicin (cf., gossypol), which is also responsible for the intercalation of doxorubicin with DNA [34,35]. At all tested solution pH values, the amine group of doxorubicin would be protonated. The electrostatic repulsion afforded at low pH between the protonated doxorubicin and PVP blocks was likely insufficient to induce complete micelle disassembly due to π-π stacking. The small amount of release observed for PEG 90 PVP 235 micelles containing doxorubicin may be due to the loss of loosely bound doxorubicin, whilst the majority was retained.
The encapsulation and release of CDKI-73 exhibited a comparatively intermediate profile, with 100% encapsulation efficiency (no release detected at pH 7.4) and an approximately linear release profile with decreasing pH, reaching an overall release of 46%. However, a lack of available data makes it challenging to relate these results to the physicochemical properties of CDKI-73. An estimated LogP value of 2.92 for CDKI-73 (Table 2) suggests that these results are consistent with its intermediate hydrophobicity (cf. doxorubicin and gossypol). Of the various functional groups in CDKI-73, only the 5-aryl-2-aminothiazole motif might be expected to have a pKa value that is of significance in this study. If it is assumed that CDKI-73 has a similar pKa value to the structural analogue, 5-phenyl-2-aminothiazole (pKa = 4.9) [36], then CDKI-73 would become protonated and positively charged in parallel with PVP protonation/ionisation. This ionisation would cause electrostatic repulsion between CDKI-73 and the PVP blocks, driving micelle disassembly and drug release in the absence of other sufficiently strong non-covalent interactions (cf. doxorubicin). The observed trends for the three drugs indicated that very hydrophobic drugs are released rapidly upon a decrease in pH due to repulsion from the hydrophilic, protonated PVP blocks, whereas more polar drugs have a stronger attraction to the protonated PVP blocks which can impede release.
To assess the effect of the f PVP on encapsulation and release, CDKI-73-loaded micelles were prepared from PEG 90 PVP 86 exhibiting a shorter PVP block length. A significant decrease in encapsulation efficiency (66%) was observed for the PEG 90 PVP 86 micelles (cf. PEG 90 PVP 235 micelles) (Figure 3), most likely because of the smaller f PVP not being sufficient to accommodate all the CDKI-73 at a loading of 10 wt%. The release profile of CDKI-73 from the PEG 90 PVP 86 micelles with decreasing pH, followed a similar trend to that observed for the PEG 90 PVP 235 micelles, with a total release of 77% at pH 4.5. This again suggested that the pH-dependent change in hydrophobicity-from hydrophobic to hydrophilic at low pH-and electrostatic repulsion between the protonated PVP blocks and protonated CDKI-73 facilitated drug release. When considering the unencapsulated portion of the drug, the pH-triggered release of encapsulated CDKI-73 from PEG 90 PVP 86 micelles was 43%, comparable with the 46% release from PEG 90 PVP 235 micelles. Doxorubicin was also successfully encapsulated in PEG 90 PVP 86 micelles (encapsulation efficiency = 100%), but no release was observed within any of the tested receiving solutions. It is likely that the observed absence of doxorubicin release was again due to the relatively strong interactions between doxorubicin and the PVP micelle core. The reduction in encapsulation efficiency and similar release profile for the PEG 90 PVP 86 polymeric micelles suggested that the f PVP predominately contributed to the loading capacity, while the underlying encapsulation and release mechanisms appeared unchanged.

Conclusions
A series of diblock PEG x PVP y copolymers were synthesised via photo-RAFT polymerisation, with varying f PVP . The PEG x PVP y copolymers exhibited pH-dependent micellisation, with D h values dependent on the f PVP . This pH-dependence is consistent with the protonation of the PVP block (pKa~5), and electrostatic repulsion induced micelle disassembly. Gossypol, CDKI-73, and doxorubicin were successfully encapsulated with >83% efficiency in PEG 90 PVP 235 micelles (10 wt% drug loading) at pH 7.4. Gossypol and CDKI-73 both exhibited favourable release profiles, with 91% and 46% released, respectively, over 9 h as the pH was decreased. In both cases, encapsulation was attributed to the hydrophobicity of the drugs and secondary interactions with the neutral PVP blocks at pH 7.4. Gossypol and CDKI-73 release at low pH was likely induced by repulsive interactions between the drugs and protonated PVP blocks and micelle disassembly. While a similar trend for CDKI-73 release was observed when encapsulated in PEG 90 PVP 86 micelles, a reduction in encapsulation efficiency indicated that the loading capacity was dependent on the f PVP . The comparatively polar doxorubicin was almost completely retained in PEG 90 PVP 86 and PEG 90 PVP 235 micelles at low pH, likely due to the presence of π-π stacking interactions with the protonated PVP blocks. These data exemplify the drug selectivity of the PVP core in PEG-b-PVP polymeric micelles, whereby the f PVP and the hydrophobicity and physiochemical properties of the drug all have a significant effect on drug encapsulation and release. This highlights the need to evaluate target drugs for their compatibility with selected pH-responsive micelle systems. Furthermore, this study indicates the potential suitability of the PEG-b-PVP system for the triggered endosomal delivery of gossypol and CDKI-73, but not the more polar doxorubicin. In addition, the micelles were appropriately sized (<130 nm) to exploit the EPR effect for uptake into cancerous tumours. Together, these data show that PEG-b-PVP polymeric micelles remain a promising means of targeted, pHresponsive drug delivery-albeit for select, compatible hydrophobic drugs-and warrant further investigation to develop and evaluate clinically relevant micelle systems.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/pharmaceutics15030977/s1. Table S1: Summary of drug standard curve results; Figure S1: Summary of GPC chromatograms of the PEG-DDMAT initiator and PEG x PVP y copolymers; Figure S2: 1 H NMR spectrum of PEG-DDMAT; Figure S3: Digital images of the 3D-printed reaction chamber; Figure S4: CAD models and drawings of the 3D-printed reaction chamber; Figure S5: 1 H NMR spectra of the PEG x PVP y copolymers; Table S2: Summary of the D h values of the PEG x PVP y micelles; Table S3: Summary of the polydispersity index values of the PEG x PVP y micelles; Figure S6: DLS particle size distributions of the PEG x PVP y micelles.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.