Chain Transfer Free Radical Polymerization Research Articles

Surface modification of MCM-41 by chain transfer free radical polymerization and their utilization for intracellular pH-responsive delivery of curcumin

In this paper, a simple method that relied on direct chain transfer free radical polymerization was reported to synthesize MSNs based polymeric composites (named as MCM-41-SH-poly (PEGMA-co-VPA)) using poly (ethylene glycol) monomethyl ether (PEGMA) and 4-vinyl phenylboric acid (VPA) as monomers. The utilization of MCM-41-SH-poly (PEGMA-co-VPA) to load and delivery curcumin (CUR) was also evaluated in detail. The structure, properties, drug loading and release behaviors were evidenced by various characterization techniques. The results from biological studies have shown that CUR can be effectively loaded on MCM-41-SH-poly (PEGMA-co-VPA) with high drug loading rate through formation of borate bond between CUR and VPA. More importantly, resultant drug-loading complexes displayed excellent water dispersibility and CUR could be released from complexes with pH responsiveness. Taken together, we have developed a simple method for surface modification MCM-41 and resultant composites could display improved properties for intracellular controlled drug delivery applications.

The Morphological and Capacitance Characteristics of A Novel Brush‐like Polymer Containing Polyaniline Segment

AbstractThe brush‐like polymer composed of poly(acrylic acid)‐block‐grafted polyaniline (PAA42‐b‐PANI30‐g‐PANIm, marked as PAA42‐PANIx) with longer conjugate chain and good capacitance characteristics was synthesized through the combination of Reversible Addition‐Fragmentation Chain Transfer (RAFT) polymerization and free radical polymerization. The structure of the obtained product was characterized with 1H‐NMR and FT‐IR. Additionally, the morphology and particle size distribution of PAA42‐PANIx ultrasonically dispersed in ethanol was characterized with transmission electron microscopy. Through cyclic voltammetry (CV), chronopotentiometry and electrochemical impedance spectroscopy (EIS), the electrochemical properties of the product were analyzed. The results showed that PAA42‐b‐PANIx was a vesicle‐like particle with smaller size, uniform size and uniform morphology distribution, it has the mean particle size of only 38 nm. The final product had good peseudocapacitance, its specific capacitance reached to 1051.51 F⋅g−1 at the current density of 3 A⋅g−1.

Controllable Preparation and Application of Quercetin Molecularly Imprinted Polymer

The quercetin molecularly imprinted polymer microspheres were prepared by reversible addition-fragmentation chain transfer free radical polymerization combined with precipitation method (RAFTPP) with the dibenzyl trithiocarbonate (DBTTC) as RAFT reagent and quercetin as template molecule. The effects of solvents and cross-linking agent on the morphology and size of polymers were investigated, and the polymers were preliminarily screened by scanning electron microscope. The adsorption experiment and adsorption model analysis were carried out on the molecularly imprinted polymers (R-MIP) prepared under optimal condition and on the molecularly imprinted polymers prepared with a traditional precipitation polymerization (T-MIP). Consequently it was determined that, QR-MIP max was 37.71 mg g−1 and QT-MIP max was 29.61 mg g−1. The results showed that the R-MIP had a good adsorptive property and was obviously superior to T-MIP. R-MIP was used as solid-phase extraction filler in combination with the HPLC method to conduct enrichment, separation and measurement of the quercetin in honeysuckle and clove leaves, which effectively removed the matrix interference and the recovery rate of this method was 93.8–102.9%.

Simple Preparation of Thiol-Ene Particles in Glycerol and Surface Functionalization by Thiol-Ene Chemistry (TEC) and Surface Chain Transfer Free Radical Polymerization (SCT-FRP).

Thiol-ene (TE)-based polymer particles are traditionally prepared via emulsion polymerization in water (using surfactants, stabilizers, and cosolvents). Here, a green and simple alternative is presented with excellent control over particle size, while avoiding the addition of stabilizers. Glycerol is applied as a dispersing medium for the preparation of off-stoichiometric TE microparticles, where sizes in the range of 40-400 µm are obtained solely by changing the mixing speed of the emulsions prior to crosslinking. Control over surface chemistry is achieved by surface functionalization of excess thiol groups via photochemical thiol-ene chemistry resulting in a functional monolayer. In addition, surface chain transfer free radical polymerization is used for the first time to introduce a thicker polymer layer on the particle surface. The application potential of the system is demonstrated by using functional particles as adsorbent for metal ions and as a support for immobilized enzymes.

Direct surface grafting of mesoporous silica nanoparticles with phospholipid choline-containing copolymers through chain transfer free radical polymerization and their controlled drug delivery

Mesoporous silica nanoparticles have attracted considerable research attention due to their various applications. Surface modification of these mesoporous silica nanoparticles with polymers not only can improve their water dispersity but can also endow several new functions, such as drug loading and delivery or targeting capability. In this work, we report a novel strategy for the direct surface grafting of phospholipid choline-containing copolymers onto Santa Barbara Amorphous-15 (SBA-15) through surface-initiated chain transfer free radical polymerization. The SBA-15 was synthesized by hydrolysis of tetraethoxysilane in the presence of poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) (P123) under acidic synthetic conditions. Next, SBA-15 was subsequently modified with thiol groups by co-condensation with γ-mercaptopropyltrimethoxysilane to obtain SBA-15-SH. Finally, the copolymers were grafted on SBA-15-SH through chain transfer free radical polymerization using 2-methacryloyloxy ethyl phosphorylcholine (MPC) and itaconic acid (IA) as monomers. The SBA-15-based polymer composites (SBA-15-SH-poly(MPC-co-IA)) were used as matrices for controlled release of cisplatin (CDDP). The data from a series of characterization techniques indicated that the monomers were successfully grafted onto SBA-15. The resultant SBA-15-SH-poly(MPC-co-IA) composites showed many remarkable physicochemical properties, such as high water dispersity, desirable biocompatibility and high drug loading capacity. These features provide the SBA-15-SH-poly(MPC-co-IA) composites with considerable potential for biomedical applications.

Development of a thiol‐ene based screening platform for enzyme immobilization demonstrated using horseradish peroxidase

Efficient immobilization of enzymes on support surfaces requires an exact match between the surface chemistry and the specific enzyme. A successful match would normally be identified through time consuming screening of conventional resins in multiple experiments testing individual immobilization strategies. In this study we present a versatile strategy that largely expands the number of possible surface functionalities for enzyme immobilization in a single, generic platform. The combination of many individual surface chemistries and thus immobilization methods in one modular system permits faster and more efficient screening, which we believe will result in a higher chance of discovery of optimal surface/enzyme interactions. The proposed system consists of a thiol-functional microplate prepared through fast photochemical curing of an off-stoichiometric thiol-ene (OSTE) mixture. Surface functionalization by thiol-ene chemistry (TEC) resulted in the formation of a functional monolayer in each well, whereas, polymer surface grafts were introduced through surface chain transfer free radical polymerization (SCT-FRP). Enzyme immobilization on the modified surfaces was evaluated by using a rhodamine labeled horseradish peroxidase (Rho-HRP) as a model enzyme, and the amount of immobilized enzyme was qualitatively assessed by fluorescence intensity (FI) measurements. Subsequently, Rho-HRP activity was measured directly on the surface. The broad range of utilized surface chemistries permits direct correlation of enzymatic activity to the surface functionality and improves the determination of promising enzyme-surface candidates. The results underline the high potential of this system as a screening platform for synergistic immobilization of enzymes onto thiol-ene polymer surfaces. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1267-1277, 2017.

Surface PEGylation of mesoporous silica materials via surface-initiated chain transfer free radical polymerization: Characterization and controlled drug release

As a new type of mesoporous silica materials with large pore diameter (pore size between 2 and 50nm) and high specific surface areas, SBA-15 has been widely explored for different applications especially in the biomedical fields. The surface modification of SBA-15 with functional polymers has demonstrated to be an effective way for improving its properties and performance. In this work, we reported the preparation of PEGylated SBA-15 polymer composites through surface-initiated chain transfer free radical polymerization for the first time. The thiol group was first introduced on SBA-15 via co-condensation with γ-mercaptopropyltrimethoxysilane (MPTS), that were utilized to initiate the chain transfer free radical polymerization using poly(ethylene glycol) methyl ether methacrylate (PEGMA) and itaconic acid (IA) as the monomers. The successful modification of SBA-15 with poly(PEGMA-co-IA) copolymers was evidenced by a series of characterization techniques, including 1H NMR, FT-IR, TGA and XPS. The final SBA-15-SH- poly(PEGMA-co-IA) composites display well water dispersity and high loading capability towards cisplatin (CDDP) owing to the introduction of hydrophilic PEGMA and carboxyl groups. Furthermore, the CDDP could be released from SBA-15-SH-poly(PEGMA-co-IA)-CDDP complexes in a pH dependent behavior, suggesting the potential controlled drug delivery of SBA-15-SH-poly(PEGMA-co-IA). More importantly, the strategy should be also useful for fabrication of many other functional materials for biomedical applications owing to the advantages of SBA-15 and well monomer adoptability of chain transfer free radical polymerization.

Preparation of PEGylated polymeric nanoprobes with aggregation-induced emission feature through the combination of chain transfer free radical polymerization and multicomponent reaction: Self-assembly, characterization and biological imaging applications

Self-assembly of amphiphilic luminescent copolymers is a general route to fabricate fluorescent polymeric microparticles (FPMs). In this work, the FPMs with aggregation-induced emission (AIE) feature were fabricated via the combination of the chain transfer free radical polymerization and “one-pot” multicomponent reaction, which conjugated the aldehyde-containing AIE active dye AIE (CHO-An-CHO) and amino-terminated hydrophilic polymer (ATPPEGMA) using mercaptoacetic acid (MTA) as the “lock” molecule. The structure, chemical compositions, optical properties as well as biological properties of the PPEGMA-An-PPEGMA FPMs were characterized and investigated by means of a series of techniques and experiments in detail. We demonstrated the final copolymers showed amphiphilic properties, strong yellow fluorescence and high water dispersibility. Biological evaluation suggested that PPEGMA-An-PPEGMA FPMs possess low cytotoxicity and can be used for cell imaging. More importantly, many other AIE active FPMs are expected to be fabricated using the similar strategy because of the good substrate and monomer applicability of the multicomponent reaction and chain transfer living radical polymerization. Therefore, we could conclude that the strategy described in this work should be of great interest for fabrication of multifunctional AIE active nanoprobes for biomedical applications.

Mussel inspired preparation of functional silica nanocomposites for environmental adsorption applications

Surface modification of nanomaterials with polymers is an effective route to render new functions and improve the performance of the final nanocomposites. Here, a facile method was developed to fabricate polyacrylic acid (PAA)-grafted monodisperse SiO2 nanoparticles (SiO2-PDA-PAA) through a combination of mussel inspired chemistry and Michael addition reaction. To obtain the products, the SiO2 nanoparticles were first coated with polydopamine (PDA) through self-polymerization of dopamine under rather mild conditions. The PDA thin films can then be further conjugated with amino-terminated PAA, which was synthesized by chain transfer free radical polymerization using cysteamine hydrochloride as a chain transfer agent and acrylic acid as a monomer. The SiO2-PDA-PAA nanocomposites were characterized via transmission electron microscopy, Fourier transform infrared spectroscopy, thermal gravimetric analysis, and X-ray photoelectron spectroscopy. The effects of contact time, solution pH, temperature and methylene blue (MB) concentration on the removal of MB were investigated. The results demonstrated that SiO2-PDA-PAA showed significant improvement in adsorption efficiency towards MB. The kinetics and isotherm studies showed that pseudo-second-order and Langmuir isotherm models were well fitted the experimental data. The values of thermodynamics parameters such as entropy change (ΔS0), enthalpy change (ΔH0) and Gibbs free energy (ΔG0) were calculated based on the Van’t Hoff equation. The negative values of thermodynamic parameters indicated that the adsorption of MB was a feasible, spontaneous and exothermic process. In summary, we developed a facile method to fabricate SiO2-based polymer nanocomposites, which showed obviously enhanced adsorption capability towards MB.

Mussel inspired preparation of MoS2 based polymer nanocomposites: The case of polyPEGMA

In this work, we report a facile strategy to prepare PEGylated MoS2 nanosheets through the combination of mussel inspired chemistry and Michael addition reaction. The MoS2 nanosheets were obtained from lithium intercalation and exfoliation method. Meanwhile, the amino-contained poly((polyethylene glycol) methyl ether methacrylate) (PPEGMA) were obtained via chain transfer free radical polymerization using cysteamine hydrochloride as the chain transfer agents and PEGMA as the monomer. To introduce PPEGMA on MoS2 nanosheets, polydopamine (PDA) thin films were first coated on the surface of MoS2 nanosheets through self polymerization of dopamine as the ad-layers, which can react with amino-terminated PPEGMA through Michael addition reaction. The structure, morphology and chemical compositions of MoS2 nanosheets and MoS2-PDA-PPEGMA have been characterized by various characterization techniques. The results demonstrated that the amino-terminated PPEGMA can be successfully immobilized on MoS2 nanosheets via PDA thin films as the ad-layers. More importantly, the strategy described in this work could also be utilized for surface immobilization of various polymers on many other materials and surfaces because of the universal adhesion of PDA and the good monomer applicability of chain transfer free radical polymerization. Taken together, we developed a facile and versatile method to fabricate multifunctional MoS2 based polymer nanocomposites with designable properties and applications via combination of mussel inspired and chain transfer free radical polymerization. The resultant composites are expected to be potentially used for drug delivery and photothermal cancer treatment.

Biomimic preparation of highly dispersible silica nanoparticles based polymer nanocomposites

Silica nanoparticles (SiO2 NPs) have been widely applied in a number of domains because of their inodorous, non-pollution, optical transparency, chemical inert and good biocompatibility. However, to achieve better performance, surface modification of SiO2 NPs to enhance their dispersibility is generally required. In this paper, a simple, facile and environmental friendly procedure was developed for surface modification of SiO2 NPs with a biocompatible polymer (polyPEGMA) via combination of mussel inspired chemistry and Michael addition reaction for the first time. Firstly, monodisperse SiO2 NPs were prepared by using a slightly modified Stöber process. Secondly, SiO2 NPs were coated with polydopamine (PDA), which was formed through self-polymerization of dopamine in an alkaline aqueous solution. Lastly, the PDA coated SiO2 NPs were facilely conjugated with polyPEGMA, which were synthesized through chain transfer free radical polymerization using cysteamine hydrochloride as chain transfer agent and poly(ethylene glycol) methyl ether methacrylate as the monomer. The successful preparation of these composites was confirmed by a number of characterization techniques including transmission electron microscopy, Fourier transform infrared spectroscopy, thermal gravimetric analysis and X-ray photoelectron spectroscopy. This method obviously enhance the dispersion of SiO2 NPs in different organic solvents and aqueous solution. This synthetic method is convenient, effective and environmental friendly, that can be also extended to modify SiO2 NPs with other functional polymers because of the features of mussel inspired chemistry and chain transfer living polymerization.

PEGylation of carbon nanotubes via mussel inspired chemistry: Preparation, characterization and biocompatibility evaluation

A novel strategy for surface modification of multi-walled carbon nanotubes (MWCNT) was developed via combination of mussel inspired chemistry and Michael addition reaction. In this procedure, pristine MWCNT were first coated with polydopamine (PDA) through self polymerization of dopamine. The PDA functionalized CNT (CNT-PDA) were further functionalized with amino-terminated polymers (polyPEGMA), which were synthesized via free radical polymerization using cysteamine hydrochloride as the chain transfer agent and poly(ethylene glycol) monomethyl ether methacylate as the monomer. The successful modification of CNT was ascertained by a series of characterization techniques including transmission electron microscopy, Fourier transform infrared spectroscopy, thermal gravimetric analysis and X-ray photoelectron spectrometry. The polymer modified CNT showed enhanced dispersibility in aqueous and organic solution. Cytotoxicity evaluation of polymers modified CNT showed that these modified CNT are biocompatible with cells. Finally, due to the universal adhesive of PDA and chain transfer free radical polymerization, this strategy developed in this work can also be extended for surface modification of many other nanomaterials with different functional polymers.

Surface modification of carbon nanotubes via combination of mussel inspired chemistry and chain transfer free radical polymerization

In this work, a novel strategy for surface modification of carbon nanotubes (CNTs) was developed via combination of mussel inspired chemistry and chain transfer free radical polymerization. First, pristine CNTs were functionalized with polydopamine (PDA), which is formed via self-polymerization of dopamine in alkaline conditions. These PDA functionalized CNTs can be further reacted with amino-terminated polymers (named as PDMC), which was synthesized through chain transfer free radical polymerization using cysteamine hydrochloride as chain transfer agent and methacryloxyethyltrimethyl ammonium chloride as the monomer. PDMC perfectly conjugated with CNT-PDA was ascertained by a series of characterization techniques including transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FT-IR), thermal gravimetric analysis (TGA) and X-ray photoelectron spectroscopy (XPS). The dispersibility of obtained CNT nanocomposites (named as CNT-PDA-PDMC) was further examined. Results showed that the dispersibility of CNT-PDA-PDMC in aqueous and organic solutions was obviously enhanced. Apart from PDMC, many other amino-terminated polymers can also be used to functionalization of CNTs via similar strategy. Therefore, the method described in this work should be a general strategy for fabrication various polymer nanocomposites.

Synthesis of ArF photoresist polymer composed of three methacrylate monomers via reversible addition-fragmentation chain transfer (RAFT) polymerization

ArF photoresist polymers were prepared via reversible addition-fragmentation chain transfer (RAFT) polymerization and free radical polymerization (FRP). Three methacrylates with lithographic functionalities including 2-ethyl-2-adamantyl methacrylate (EAdMA), α-gamma-butyrolactone methacrylate (GBLMA), and 3-hydroxy-1-adamantyl methacrylate (HAdMA), were used as monomer components and 2-cyanoprop-2-yl-1-dithionaphthalate (CPDN) was used as the chain transfer agent (CTA). In both polymerizations, the order of monomer reactivity was GBLMA>HAdMA>>EAdMA. This caused a composition gradient in RAFT polymerization as well as composition inhomogeneity in FRP. The polymers prepared by RAFT polymerization had lower molecular weight distributions and more uniform compositions. The improvement in molecular weight distribution and composition uniformity of the polymers prepared by RAFT polymerization should be beneficial for the ArF lithography process.

Synthesis and Association Behavior of Telechelic Poly(N-isopropylacrylamides) with Azobenzene End Groups

A telechelic poly(N-isopropylacrylamide) (PNIPAM) with azobenzene end-groups (telechelic Az-PNIPAM) was prepared by reversible addition-fragmentation chain transfer (RAFT) free radical polymerization of NIPAM. This polymer self-assembles in cold water forming nanoparticles with a hydrodynamic radius (R H) of 8 nm and an aggregation number of 29 chains. The thermoresponsive behavior of Az-PNIPAM in water was investigated by turbidimetry and light scattering. In addition, the photoresponsive behavior of aqueous Az-PNIPAM was investigated by monitoring the changes with temperature of the solution and transmittance at 650 nm before and after irradiation at 366 nm.

Selective endothelial cell attachment to peptide-modified terpolymers

In a previous report we screened a combinatorial peptide library to identify novel ligands that bind with high affinity and specificity to human blood outgrowth endothelial cells (HBOEC). In this study we demonstrate the use of the phage display-selected-HBOEC-specific peptides as a tool to direct and modulate endothelial cell (EC) behavior with a focus on designing functional biomaterials intended for use in cardiovascular applications. First, we ensured that our peptide ligands did not interfere with EC function as tested by proliferation, migration, tube formation, and response to vascular endothelial growth factor. Second, peptides that supported EC function were incorporated into methacrylic terpolymers via chain transfer free radical polymerization. The HBOEC-specific peptide, TPSLEQRTVYAK, when covalently coupled to a terpolymer matrix, retained binding affinity towards HBOEC in a serum-free medium. Under the same binding conditions, the attachment of human umbilical vein endothelial cells (HUVEC) was limited, thus establishing HBOEC specificity. To our knowledge, this is the first report demonstrating specificity in binding to peptide-modified biomaterials of mature EC, i.e., HUVEC, and EC of progenitor origin such as HBOEC. The findings from this work could facilitate the development of autologous cell therapies with which to treat cardiovascular disease.

One-step copolymerization modified magnetic nanoparticles via surface chain transfer free radical polymerization

Copolymer brushes growing onto magnetic nanoparticles were prepared by surface chain transfer free radical polymerization. Block copolymer brushes (P(PEGMA)- co-PNIPAAm) consist of poly(ethylene glycol) monomethacrylate (PEGMA) and N-isopropylacrylamide monomer. X-ray photoelectron spectroscopy (XPS) characterized the chemical composition of copolymer. Thermogravimetric analysis (TGA) suggested that the amount of copolymer on magnetic nanoparticles decreased with increasing azodiisobutyronitrile (AIBN). The saturation magnetization decreased significantly with increasing P(PEGMA)- co-PNIPAAm. The thermosensitive point is about 43 °C for magnetic nanoparticles with 33.8% P(PEGMA)- co-PNIPAAm.

Amphiphilic telechelic poly(N-isopropylacrylamide) in water: From micelles to gels⋆

We report the first study of aqueous solutions (0.025 gL(-1) to 46 gL(-1)) of a telechelic poly(N-isopropylacrylamide) with octadecyl termini (C(18)-PNIPAM-C(18), M(w): 37000, 320 NIPAM units, M(w)/ M(n)=1.07) obtained by reversible addition-fragmentation chain transfer (RAFT) free radical polymerization of N-isopropylacrylamide. Static and dynamic light scattering measurements and fluorescence spectroscopy, using 8-anilino-1-naphthalenesulfonic acid (ANS) as probe, yielded the concentration dependence of the size and aggregation number of C(18)-PNIPAM-C(18) micelles in cold ( 20( degrees )C) dilute aqueous solutions. Concentrated solutions ( c>20 gL(-1)) form transient gels exhibiting an oscillatory shear behavior that can be approximated by a single-relaxation Maxwellian model. Aqueous solutions of C(18)-PNIPAM-C(18) undergo a phase transition upon heating to 31.5( degrees )C as determined by microcalorimetry. The heat-induced phase separation of dilute (0.025 gL(-1)) C(18)-PNIPAM-C(18) solutions yields a fluid that is colloidally stable at temperatures higher than 33( degrees )C. The overall results are consistent with a model assuming the formation of flowerlike micelles in the dilute regime and a network of micelles connected by telechelic chains in the concentrated regime.

Synthesis of starlike N-(2-hydroxypropyl)methacrylamide copolymers: potential drug carriers.

Starlike HPMA copolymers were synthesized by conjugating semitelechelic poly[N-(2-hydroxypropyl)-methacrylamide] macromolecules (ST-PHPMA, arm) with PAMAM dendrimers (core: G2, G3, G4). ST-PHPMA was synthesized by chain transfer free radical polymerization, and the terminal -COOH was activated with N-hydroxysuccinimide. Doxorubicin (DOX) was introduced into the starlike HPMA copolymer to evaluate its potential as a drug delivery system. The polymers were characterized with SEC, NMR, and UV. Cytotoxicity of the DOX containing starlike HPMA copolymer was determined on an A2780 human ovarian carcinoma cell line and compared with DOX-containing linear HPMA copolymers. The rate of in vitro DOX release from polymer--DOX conjugates in the presence of cathepsin B (CP-B, lysosomal cysteine proteinase) was determined and correlated with cytotoxicity results.

Functionalized semitelechelic poly[N-(2-hydroxypropyl)methacrylamide] for protein modification.

Semitelechelic poly[N-(2-hydroxypropyl)methacrylamide]s (ST-PHPMA) with different functional end groups, namely carboxyl, methyl ester, hydrazide, and amino groups, were prepared by chain transfer free-radical polymerization. 2,2'-Azobisisobutyronitrile (AIBN) was used as an initiator and 3-mercaptopropionic acid, methyl 3-mercaptopropionate, 3-mercaptopropionic hydrazide, and 2-mercaptoethylamine were used as chain-transfer agents. The semitelechelic polymers have been characterized by end-group analysis, size-exclusion chromatography (SEC), and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The effects of the concentrations of the mercaptans and the initiator on the molecular weight of the polymers have been investigated. The higher the concentration of mercaptan, the lower the molecular weight of ST-PHPMA. The concentration of initiator did not have a significant effect on the molecular weight of the semitelechelic polymers. The end groups of the ST polymers can be readily transformed by polymeranalogous reactions. A model protein, alpha-chymotrypsin, has been modified with ST-PHPMA-CONHNH2 and ST-PHPMA-COOSu and the conjugates characterized by MALDI-TOF MS. The activity of modified chymotrypsins toward a high molecular weight substrate, P-Gly-Leu-Phe-NAp (where P is the HPMA copolymer backbone, and NAp is p-nitroanilide), was slightly lower than the activity of the native enzyme. The cleavage of a low molecular weight substrate, Z-Gly-Leu-Phe-NAp, by modified chymotrypsins was dependent on their structure. Whereas the activity of the amino group modified chymotrypsins was higher than that of the native enzyme, the activity of carboxyl-modified chymotrypsins was lower than that of the native enzyme. In summary, the data seem to indicate that ST-PHPMA is an effective protein-modifying agent.