Polymer Masked−Unmasked Protein Therapy. 1. Bioresponsive Dextrin−Trypsin and −Melanocyte...

Polymer Masked-Unmasked Protein Therapy. 1. BioresponsiveDextrin-Trypsin and -Melanocyte Stimulating Hormone

Conjugates Designed for r-Amylase Activation

Ruth Duncan,*,† Helena R. P. Gilbert,† Rodrigo J. Carbajo,‡ and María J. Vicent†,§

Centre for Polymer Therapeutics, Welsh School of Pharmacy, Cardiff University, King Edward VII Avenue,Cardiff, CF10 3XF, U.K., and Polymer Therapeutics Laboratory and Laboratory of Structural Biology,

Centro de Investigación Príncipe Felipe, Av. Autopista del Saler 16, E-46012 Valencia, Spain

Received September 26, 2007; Revised Manuscript Received December 12, 2007

Polymer-protein conjugation, particularly PEGylation, is well-established as a means of increasing circulationtime, reducing antigenicity, and improving the stability of protein therapeutics. However, PEG has limitationsincluding lack of polymer biodegradability, and conjugation can diminish or modify protein activity. The aim ofthis study was to explore a novel approach for polymer-protein modification called polymer-masking-unmasking-protein therapy (PUMPT), the hypothesis being that conjugation of a biodegradable polymer to a protein wouldprotect it and mask activity in transit, while enabling controlled reinstatement of activity at the target site bytriggered degradation of the polymeric component. To test this hypothesis, dextrin (R-1,4 polyglucose, a naturalpolymer degraded by R-amylase) was conjugated to trypsin as a model enzyme or to melanocyte stimulatinghormone (MSH) as a model receptor-binding ligand. The effect of dextrin molecular weight (7700, and 47200g/mol) and degree of succinoylation (9–32 mol %) on its ability to mask/unmask trypsin activity was assessedusing N-benzoyl-L-arginine-p-nitroanilide (L-BAPNA). Dextrin conjugation reduced enzyme activity by 34–69%depending on the molecular weight and degree of succinoylation of dextrin. However, incubation with R-amylaseled to reinstatement of activity to a maximum of 92–115%. The highest molecular dextrin (26 mol % succinoylation)gave optimum trypsin masking-unmasking. This intermediate was used to synthesize a dextrin-MSH conjugate(dextrin Mw ) 47200 g/mol; MSH content 37 wt %), and its biological activity ((R-amylase) was assessed bymeasuring melanin production by murine melanoma (B16F10) cells. Conjugation reduced melanin production to11%, but addition of R-amylase was able to restore activity to 33% of the control value. These were the firststudies to confirm the potential of PUMPT for further application to clinically important protein therapeutics.The choice of masking polymer, activation mechanism, and the rate of unmasking can be tailored to therapeuticapplication.

Introduction

Although the number of antibody and protein therapeuticsentering routine clinical use is increasing exponentially,1,2 thereare still significant challenges for protein formulation and clinicaluse. These include poor solubility, susceptibility to chemicaland proteolytic inactivation, and suboptimal pharmacokineticsat the whole body level (due to rapid reticuloendothelial system(RES) clearance and, for small proteins and peptides, rapid renalelimination) and the cellular level (inability to reach intracellularpharmacological targets). Additionally, some proteins can beantigenic causing anaphylactic reactions. Polymer-proteinconjugation can be used to overcome these problems andpoly(ethylene glycol) (PEG) conjugation (PEGylation; reviewedin refs 3 and 4) has been particularly successful. PEGylation(reviewed in ref 5) has generated novel treatments for cancer(e.g., Oncaspar, Neulasta), viral diseases (e.g., PEGASYS,PEGINTRON), acromegaly (Pegvisomant), and age-relatedmacular degeneration (MACUGEN). Despite the ability ofcontemporary PEGylation chemistry to give well-defined con-

jugates (reviewed in ref 6) and to introduce biodegradablePEG-protein linkers,7 the use of PEG does have disadvantages,not least its lack of biodegradability which brings the risk ofpolymer accumulation following chronic administration.

The aim of this study was to develop a novel approach forpolymer–protein modification, called polymer masking–unmasking protein therapy (PUMPT). It was hypothesized thatcoupling a biodegradable polymer to a protein would create aconjugate that would be inactivate in transit due to proteinmasking but, by triggered degradation of the polymer, wouldreinstate protein activity at a rate tailored to suit its mode ofaction. Additionally, careful choice of polymer and the activatingmechanism may allow preferential localization of pharmacologi-cal activity to the desired target for action (summarized in Figure1). To test the feasibility of PUMPT the polysaccharide dextrin,R-1,4-poly(glucose) was selected as the first model polymer(preliminary data were reported in refs 8 and 9). Dextrin isapproved for clinical use as the peritoneal dialysis solution(Icodextrin),10 as a solution to prevent postoperative adhesions(Adept),11 and as a formulation solution for peritoneal admin-istration of 5-fluorouracil.12 Thus its clinical safety is welldocumented.13 Degradation to maltose and isomaltose is medi-ated by R-amylase, and we have previously shown that dextrinmodification by succinoylation14 can be used to introduce thereactive groups necessary for covalent drug conjugation (e.g.,to doxorubicin15 or amphotericin B16). Moreover, the degree

* Corresponding author: tel, 029 20876419; fax, 029 20874536; e-mail,[email protected].

† Centre for Polymer Therapeutics, Welsh School of Pharmacy.‡ Laboratory of Structural Biology, Centro de Investigación Príncipe

Felipe.§ Polymer Therapeutics Laboratory, Centro de Investigación Príncipe

Felipe.

Biomacromolecules 2008, 9, 1146–11541146

10.1021/bm701073n CCC: $40.75 2008 American Chemical SocietyPublished on Web 03/19/2008

of succinoylation can be used to tailor amylase degradationrate.17 Trypsin was used as a model enzyme, and melanocytestimulating hormone (MSH) as a model receptor binding ligand.

The effect of dextrin molecular weight (7700, and 47200g/mol) and degree of succinoylation (9–32 mol %) on its abilityto mask/unmask trypsin activity was first assessed. Conjugateswere characterized by NMR, FTIR, gel permeation chromatog-raphy (GPC), SDS PAGE, and circular dichroism (CD) spec-troscopy, and the biological activity of trypsin conjugates wasassessed (R-amylase using N-benzoyl-L-arginine-p-nitroanilide(L-BAPNA) as a substrate. The dextrin-MSH conjugate wassynthesized using dextrin of Mw ) 47200 g/mol (26 mol %succinoylation). In this case biological activity ((R-amylase)was assessed by measuring stimulation of melanin productionin B16F10 murine melanoma cells.

Materials and Methods

Materials. A 40% solution of acrylamide/bis(acrylamide) (ratio37.5:1),ammonium persulfate, salivary R-amylase, L-BAPNA, biocincho-ninic acid (BCA) solution, blue dextran, bromothymol blue, copper(II)sulfate, dextrin (type 1 from corn, Mw 7700 g/mol), anhydrous N,N-dimethylformamide (DMF), anhydrous succinic anhydride,tris(hydroxymethyl)aminomethane HCl (trizma HCl), trypan bluesolution (0.4% w/v), MSH, and porcine pancreatic trypsin (type 1X-S)were from Sigma-Aldrich (U.K.). Pullulan gel filtration standards (Mw

) 5800–186000 g/mol) were from Polymer Laboratories (U.K.).Bromophenol blue, coomassie brilliant blue G-250, 2-mercaptoethanol,sodium dodecyl sulfate (SDS), prestained SDS-PAGE standards, andN,N,N,N′-tetramethylethylenediamine (TEMED) were from Bio-Rad(USA). 4-Dimethylaminopyridine (DMAP) was from Fisher Scientific(U.K.). Glycine (electrophoresis grade) was from ICN Biomedicals(USA), and N-hydroxysulfosuccinimide (sulfo-NHS) and N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide (EDC) were from Pierce Chemi-cal Co. (USA). All solvents were of general reagent grade (unless stated)and were from Fisher Scientific (U.K.). Samples were dialysed usingSpectra/Por Membranes (molecular weight cutoff 25000 or 2000 g/molfrom BDH Merck (U.K.)).

The B16F10 murine melanoma cell line was from the Americantype culture collection (ATCC) (USA). RPMI 1640 with phenol red(PR), glutamax, fetal bovine serum (FBS), and trypsin–ethylenediamine-tetraacetic acid (EDTA) (0.05% w/w trypsin, 0.53 mM EDTA) wereall from Invitrogen Life Technologies (U.K.). CO2 and N2 (medicalgrade, 95% v/v) and liquid N2 were supplied by BOC (U.K.).

Synthesis and Characterization of Succinoylated Dextrin. Suc-cinoylated dextrins (9–32 mol % carboxyl groups) were prepared aspreviously described by Hreczuk-Hirst et al.14 (Scheme 1a). Briefly,

Figure 1. Concept of PUMPT showing masking and then theoreticalregeneration of protein activity in the presence of activating enzyme.

Scheme 1. Synthesis of Dextrin-Trypsin Conjugates

Table 1. Characteristics of Succinoylated Dextrins and Conjugates

dextrins dextrin-trypsin conjugates

compound Mwa (g/mol) (Mw/Mn) succinoylation (mol %) Mw

a (g/mol) (Mw/Mn) trypsin content (wt %)

trypsin 23400dextrin 7700 (1.4)succ-dextrin (7700 g/mol) 14600 (1.3) 15 25700 (1.7) 72

55000b

succ-dextrin (7700) 13500 (1.4) 23 14000 (1.5) 6739300b

succ-dextrin (7700) 15500 (1.5) 32 19800 (1.5) 8147700b

dextrin 47200 (1.5)succ-dextrin (47200) 61300 (1.4) 9 84400 (1.6) 51

110600b

succ-dextrin (47200) 57800 (1.4) 18 101800 (1.6) 78122900b

succ-dextrin (47200) 39300 (1.4) 26 49800 (1.3) 7648200b

a Mw estimated by GPC using pullulan standards. b Mw estimated by GPC using protein standards.

Dextrin-Protein Conjugates Biomacromolecules, Vol. 9, No. 4, 2008 1147

with dextrin of Mw 47200 g/mol, 9 mol % succinoylation was achievedas follows. Dextrin (1 g, 2.12 × 10-5 mol) and DMAP (30 mg, 2.41× 10-4 mol) were placed in a sealed flask and dissolved in anhydrousDMF (10 mL) before purging with nitrogen. Succinic anhydride (70mg, 7 × 10-4 mol) dissolved in anhydrous DMF (2.5 mL) was thenadded. The reaction was left for 16-18 h (overnight) at 50 °C undernitrogen, and then DMF was partially evaporated under reducedpressure. The product was purified by precipitation into rapidly stirringdiethyl ether (∼250 mL) and allowed to stir for 4 h before the solutionwas filtered under vacuum. The solid obtained was redissolved in doubledistilled water (10 mL) and further purified by dialysis (molecular

weight cutoff 2000 g/mol) against double distilled water (6 × 5 L)over 48 h. The solution was finally freeze-dried to yield succinoylateddextrin product as a white solid (∼60% yield) that was characterizedby FTIR (Avatar 360 ESP spectrometer with EZ OMNIC ESP 5.2software; Thermo Nicolet, Loughborough, U.K.) and 1H NMR toconfirm identity, titration to determine the carboxyl group content, andby GPC to measure the approximate molecular weight and polydis-persity. Titrimetric analysis was conducted using NaOH (5 × 10-4

M) as base and bromothymol blue (1% in ethanol) as an indicator.Samples for GPC were prepared in PBS (3 mg/mL), and maltose (Mw)342 g/mol) was used to mark the Vb and blue dextran (Mw ) 2000000g/mol) to mark the Vo. Pullulan standards were used as a reference.

Synthesis and Characterization of Dextrin–Trypsin Conju-gates. Succinoylated dextrins (Mw ∼ 7700 and 47200 g/mol, 9–32 mol% succinoylation) were then conjugated to trypsin,8,9,14 (Scheme 1b).Briefly, EDC (77 mg, 4.00 × 10-4 mol) was added as solid to a watersolution (8 mL) of succinoylated dextrin (100 mg, 1.657 × 10-1 molof COOH groups) and the reaction mixture stirred for 10 min at roomtemperature. Sulfo-NHS (88 mg, 4.04 × 10-4 mol) was added also asa solid, and the reaction continued with stirring for a further 40 min atroom temperature. Trypsin (198 mg, 8.29 × 10-6 mol) dissolved indouble distilled water (5 mL) was added dropwise and the pH of thereaction adjusted to ∼8 with 1 M NaOH. The reaction was allowed tocontinue for 16 h at room temperature. The precipitated urea was filteredoff, and the conjugate solution was purified by dialysis (molecularweight cutoff 25000 g/mol) against double distilled water (5 L) over48 h. After freeze-drying dextrin-trypsin conjugate was obtained as awhite solid (yield ∼50%).

Dextrin-trypsin conjugates were characterized by GPC (TSKG4000PWXL and G3000 PWXL columns in series, mobile phase PBS(0.1 M, pH 7.4), flow rate of 1 mL/min) to establish approximatemolecular weight and purity (against pullulan and protein standards),the BCA assay to determine protein content, and by SDS-PAGE toestablish purity. The BCA assay18 used trypsin standards. SDS-PAGEelectrophoresis was used to assess levels of free protein. SDS gels(12.5%) were prepared to 0.75 mm thickness. Conjugate samples (1mg/mL in dd H2O) and reference samples (trypsin and a mixture ofdextrin and trypsin) were prepared by diluting (1:1) with denaturingsolution (3.8 mL of H2O, 5 mL; 0.5 M Tris HCl (pH 6.8), 8 mL 10%w/v SDS, 4 mL of glycerol, 2 mL of 2-mercaptoethanol, 0.4 mL ofbromophenol blue 1% w/v) and heated for 5 min at 100 °C. Thesesamples and protein molecular weight markers were loaded (10 µL),and the gel was run for 1 h at 200 V. It was then rinsed and stained (1h) with coomassie blue stain before clearing by soaking in destainingbuffer (35% methanol/5% acetic acid) for 1 h and then rehydrating in5% methanol/7% acetic acid. Finally, the gel was rinsed with distilledwater and dried for 2 days.

Characterization of Dextrin-Trypsin Conjugates by NMRand Circular Dichroism Spectroscopy. NMR Spectroscopy. NMRspectra were recorded at 27 °C on a Bruker Avance Ultrashield Plus600 spectrometer equipped with 5 mm single-axis gradient TCIcryoprobe. Data were processed using the program Topspin 1.3 (BrukerGmbH, Karlsruhe, Germany). The samples were prepared to aconcentration of 0.22 mM in 90/10% H2O/D2O.

One-Dimensional 1H Experiments. Proton NMR spectra wereacquired with 16K complex points and a spectral width of 8400 Hz.The total number of scans was 32, with a repetition delay of 1.2 s. AWATERGATE scheme was used to suppress the water signal.

Diffusion Experiments. Pulsed field gradient NMR spectroscopy wasused to measure translational diffusion by fitting the integrals orintensities of the NMR signals to the following equation

I) I0 exp[-Dγ2g2δ2(∆- δ ⁄ 3)] (1)

where I is the observed intensity, I0 the reference intensity (unattenuatedsignal intensity), D the diffusion coefficient, γ the gyromagnetic ratioof the observed nucleus, g the gradient strength, δ the length of thegradient, and ∆ the diffusion time. Two-dimensional diffusion-ordered

Figure 2. Characterization of succinolylated dextrin and dextrin-trypsinconjugates. (a) FTIR spectra showing the increase in peak intensityat 1720 cm-1 with increasing incorporation of carboxyl groups. (b)FPLC traces of a dextrin-trypsin conjugate prepared with dextrinhaving different degrees of succinoylation. (c) SDS page electro-phoresis of dextrin-trypsin conjugates showing absence of freetrypsin.

1148 Biomacromolecules, Vol. 9, No. 4, 2008 Duncan et al.

NMR spectroscopy (DOSY) was performed with a stimulated echosequence using bipolar gradient pulses19 and with a WATERGATEscheme to suppress the water signal. The lengths of pulses and delayswere held constant, and 20 spectra of 32 scans each were acquiredwith the strength of the diffusion gradient varying between 5% and100%. The lengths of the diffusion gradient and the stimulated echowere optimized for each sample. Typical values were δ ) 5–7 ms, ∆) 50–150 ms. Processing and analysis of the data were performed withthe DOSY protocol included in the Topspin 1.3 software package.

Circular Dichroism (CD) Measurements. CD spectra of dextrin (Mw

47200 g/mol), succinoylated dextrin (Mw 47200 g/mol; 26 mol %),trypsin, succinoylated dextrin (Mw 47200 g/mol; 26 mol %) plus trypsin,and the dextrin-trypsin conjugate were acquired on a Jasco-810spectropolarimeter equipped with a Peltier temperature control in quartzcells of 0.1 cm path length at two different temperatures (4 and 37 °C)and in two different buffers (Tris at pH 8.2 (40 mM Tris, 16 mM CaCl2)and H2O at pH 5.5) trying to mimic biological conditions. Compoundswere analyzed at 2 mM trypsin-equiv in all cases; when controlpolymers were studied, the same amount of polymeric carrier as indextrin-trypsin conjugate was used. CD spectra were the average of40 scans made at 0.2 nm intervals, and always the same buffer withoutcompounds, used as baseline, was subtracted. Results are expressed asmean molar residue ellipticities [Q]MR (deg cm2/dmol).

Degradation of Dextrin and Succinoylated Dextrins byAmylase.17 Dextrin (Mw ) 7700 and 47200 g/mol) or succinoylateddextrins (47200 g/mol and ∼15 and ∼30 mol % succinoylation) weredissolved in PBS (4 mL, pH 7.4) at a concentration of 3.75 mg/mL.Amylase (400 µL; 2.5 units/mL) was added and the mixture incubatedat 37 °C for 5 h. At various times, samples (100 µL) were taken,immediately snap frozen in liquid nitrogen to stop the reaction, andstored at –20 °C until analysis. Before GPC analysis, samples wereplaced for 5 min in a boiling water bath to denature enzyme activityand precipitate the protein. The supernatant was then analyzed by GPCto determine the change in molecular weight of the sample over time.

Analysis of Trypsin Activity.20 L-BAPNA (7 mg/mL stock inDMSO) and the trypsin and dextrin-trypsin conjugate samples (0.1mg/mL stock in Tris buffer; pH 8.2, 40 mM Tris, 16 mM CaCl2) wereprepared. L-BAPNA to give a final concentration in the range 1.61 ×10-4 to 9.66 × 10-4 M was added to a 1 mL cuvette followed bytrypsin (200 ng or 2 µg) or dextrin-trypsin conjugates (200 ng or 2µg of trypsin-equiv) to give a total volume of 1 mL. The release ofp-nitroanilide (NAp) (ε410 nm ) 8800 L/mol per cm) was then measuredat 37 °C for 20 min at 400 nm and NAp released expressed as apercentage of that seen for native trypsin. The data obtained were alsotransformed using a Hanes-Woolfe plot and Kcat calculated.21

To study the effect of R-amylase on the activity of the dextrin-trypsinconjugates, they (300 µL of 1 mg/mL stock) were first incubated withR-amylase (120 µL of 1 mg/mL stock) for 16 h at 37 °C in Tris buffer(2580 µL, pH 8.2). Then samples (2 µg trypsin-equiv) were added toa 1 mL cuvette containing L-BAPNA (concentrations from 1.61 × 10-4

to 9.66 × 10-4 M) and NAp release measured at 400 nm as describedabove.

Synthesis of Dextrin-MSH Conjugates. Dextrin (47200 g/mol; 26mol % succinoylation) was conjugated to MSH as described above

with minor modification. Briefly, 30 mg (5.15 × 10-7 mol) succinoy-lated dextrin was reacted with 5 mg (2.6 × 10-6 mol) of MSH for16 h at room temperature. The resulting dextrin-MSH conjugates werepurified by FPLC fractionation (Superdex HR 10/30; mobile phase PBS0.1 M (pH 7.4) and a flow rate of 0.5 mL/min), desalted using vivaspincentrifugal filters and lyophilized. Total MSH content was determinedusing the BCA assay18 (with an MSH standard curve), and purity wasassessed using SDS-PAGE electrophoresis and analytical FPLC.

In addition, degradation of dextrin-MSH conjugate, dextrin, andsuccinoylated dextrin (all 1 mg/mL) was monitored during incubationin tissue culture media (RPMI + 10% FBS or RPMI + 10% heatinactivated FBS diluted 1:20 in PBS; pH 7.4). Aliquots (200 µL)of incubation medium were withdrawn at t ) 0 and 16 h and snapfrozen in liquid nitrogen before GPC analysis (TSK-gel columns G4000PWXL and G3000 PWXL in series). The change in molecular weightwas expressed as a percentage of the starting molecular weight of thesample.

Melanin Production in B16F10 Cells Incubated with MSHand Dextrin-MSH. B16F10 cells were routinely maintained in RPMI1640 (+ Glutamax) media + 10% FBS and split twice weekly at aratio of 1:9. Because of the complexity of this assay, it was firstnecessary to conduct a number of preliminary experiments to optimizethe assay conditions. As was shown by O’Hare et al.,22 it was foundhere that MSH-induced melanin production decreased with increasingtime after B16F10 subculture (results not shown), and the optimumtime for MSH addition was 24 h after subculture. Melanin productiondecreased as incubation time increased, reaching a plateau after 72 h.This might have been due to receptor down-regulation and/or mediumdepletion of the substrate tyrosine.23 An optimized assay used todetermine the ability of dextrin-MSH conjugates ((R-amylase; 0.2mg/mL; 10 ui/mL) to induce melanin production was as follows.Twenty four hours after cell seeding (2.5 × 104 cells/mL) into a 96well plate, the incubation media was removed and MSH or dextrin-MSH(10-4M MSH-equiv) dissolved in RPMI + 10% heat-inactivated FBSand sterile filtered (0.2 µm) was added (100 µL). The cells were thenincubated for 72 h before absorbance at 405 nm was measured usinga Tecan plate reader to quantify melanin production. Values wereexpressed as a percentage of the free MSH control.

Results

Dextrin Conjugates, Synthesis and Characterization. Thecharacteristics of the library of succinoylated dextrins synthe-sized are summarized in Table 1. 1H NMR, 13C NMR and FTIRconfirmed succinoylation, and the FTIR peak seen at 1720 cm-1

increased with increasing modification (Figure 2a) (also see ref14). Titration gave dextrin succinoylation values of between 9and 32 mol % (Table 1), and typically the reaction conversionefficiency was 70–100%. GPC suggested an increase in dextrinmolecular weight following succinoylation (Mw of ∼12000–16000g/mol and ∼60000 g/mol) without any change in polydispersity(1.3–1.5) (Table 1), but this likely reflects only a difference inpolymer coil conformation.

Table 2. Activity of Dextrin-Trypsin Conjugates ((R-amylase)

dextrin Mwa(g/mol)

degree ofsuccinoylation (mol %)

proteincontent (wt %)

remainingactivity (%)

amylase restoredactivity (%)

Kcat (s-1)

unmaskedmasked

dextrin-trypsin conjugates7700 15 72 55 67 4.09 ( 2.19 1.27 ( 0.057700 23 67 69 52 2.54 ( 1.78 1.26 ( 0.577700 32 81 51 92 2.59 ( 0.66 2.30 ( 0.5447200 9 51 63 115 2.69 ( 0.25 2.49 ( 0.3147200 18 78 46 64 2.23 ( 0.78 2.40 ( 0.7147200 26 76 34 58 1.25 ( 0.38 2.49 ( 0.33

trypsin 2.52–2.93a Mw was estimated by GPC using pullulan standards.


Using the succinoylated dextrin intermediates, dextrin-trypsinconjugates were prepared and their characteristics are sum-marized in Tables 1 and Table 2. The trypsin content was 50–80wt % (neither dextrin nor succinoylated dextrin interfered withthe BCA protein assay, results not shown). Both GPC (notshown) and FPLC analysis (Figure 2b) indicated no free trypsinin conjugates produced from the higher molecular weightintermediate with up to 26 mol % succinoylation, and this wasconfirmed for both families of conjugate by SDS-PAGEelectrophoresis (Figure 2c). However, there was some freetrypsin in the lower molecular weight dextrin conjugates. GPCestimation of molecular weight characteristics was made usingboth pullulan and protein standards knowing that neither wouldgive an absolute molecular weight but only some indication of

hydrodynamic volume. Apparent molecular weights were∼14000–102000 g/mol depending on the starting molecularweight of the dextrin used and polydispersity by GPC was1.3–1.6 (Table 1). SDS page electrophoresis, however, showedthat a broad range of conjugates were present (Figure 2c).

Dextrin-trypsin conjugate formation was confirmed bycomparing the 1H NMR spectra acquired for trypsin, succinoy-lated dextrin, the dextrin-trypsin conjugate and an equimo-lecular mixture of succinoylated dextrin and trypsin (Figure 3).A close look at the aromatic/amide proton region of the 1H NMRspectra (6–11 ppm) shows a significant difference between thethree samples, with the profile of the NH region of trypsin(Figure 3a) being dramatically different from that of the putativeconjugate (Figure 3d) indicating that the protein is surroundedby a completely different chemical environment. Noncovalentpolymer encapsulation/interaction with trypsin can be discountedby comparing the spectra for trypsin, conjugate, and the simplemixture of succinoylated-dextrin plus trypsin. In the latter case(Figure 3c), the 1H signals from the amide/aromatic region showsmall changes when compared with the trypsin spectrum, thechemical shift values being maintained although affected by ageneral broadening, probably due to unspecific interactions ofthe protein with the polymer.

DOSY identifies species depending on their molecular size/hydrodynamic radius.24 The spectrum of the succinoylateddextrin-trypsin conjugate gives valuable qualitative informationabout the number of species present in solution and shows atleast three diffusion coefficients (Figure 3e, dashed lines). Fromthe 1H projection on the F2 axis it can be concluded that twoof the species contain trypsin as they show cross peaks in theNH/aromatic region for proteins (above 6 ppm). As the SDS-PAGE analysis of the same sample shows no free trypsinpresent, these two diffusion coefficients must correspond to twopolymer-protein conjugates. The remaining species only cor-relates with 1H signals located in the region of the polymermoiety (2.5–5.5 ppm) and therefore could be ascribed to theremaining free polymer.

CD spectroscopy (Figure 4) also showed clear differencesbetween the dextrin-trypsin conjugate and the simple mixtureof the two components. In good agreement with the NMR data,the presence of free succinoylated dextrin in the sample seemedto result in nonspecific interactions with the protein chain leadingto a greater structural mobility and loss of ∼10% of the trypsinR-helix conformational parameter. This was not seen in theconjugate sample, as it displayed a spectrum very similar tothat of trypsinsanother clear proof of protein conjugation tothe polymeric carrier. As expected, dextrin and succinoylateddextrin showed a complete unstructured conformation.

GPC evaluation of dextrin and succinoylated dextrin degrada-tion by R-amylase revealed a decrease in molecular weight withtime (Figure 5). The extent of degradation of native dextrins ofstarting molecular weight of 47200 and 7700 g/mol at 5 h was95% and 30%, respectively. The succinoylated dextrins weredegraded more slowly than parent polymer, the higher thesuccinoylation, the slower the degradation rate. Using theseincubation conditions a plateau in molecular weight was reachedin all cases.

Dextrin-Trypsin Activity ((r-Amylase). When comparedat the same protein concentration, the activity of the dextrin–trypsin conjugates was reduced compared to free trypsin in allcases (Figure 6; Table 2). For those conjugates prepared from47200 g/mol dextrin, enzyme activity fell significantly from 63to 34% in relation to an increase (from 9 to 26 mol %) in thedegree of intermediate succinoylation. In contrast, for those

Figure 3. 1H NMR and DOSY spectra of trypsin, dextrin-trypsin, amixture of succinoylated dextrin and trypsin, and succinoylateddextrin. (Prepared using succinoylated dextrin (47200 g/mol; 26 mol%)). Expansion of the NH/aromatic region from the 1D 1H spectraare shown for (a) trypsin, (b) succinoylated dextrin, (c) a mixture ofsuccinoylated dextrin and trypsin, and (d) dextrin-trypsin conjugate.(e) Diffusion DOSY spectrum for the dextrin-trypsin conjugatesample. Typical experimental parameters: diffusion time 50 ms,gradient time 5 ms, gradient strength 5–100%. The horizontal axisprojection shows the 1H spectrum of the corresponding sample.


conjugates synthesized using the lower molecular weight dextrin,the degree of succinoylation did not correlate with retainedenzyme activity. This was always in the range 51–69%.Calculation of the rate constants Km, Vmax, and Kcat showedthat conjugation of trypsin to dextrin typically did not alter Km,but the Vmax was reduced ∼10-fold. The Kcat values calculatedtypically were 2–3 s-1(Table 2).

The enzyme activity of conjugates exposed to R-amylase wasusually increased (Figure 7, Table 2). For example, for the lowermolecular weight dextrin modified to 32 mol %, trypsin activitywas restored to 92% of the control following R-amylaseexposure. In the case of the higher molecular weight dextrinhaving 26 mol % succinoylation, R-amylase exposure increasedtrypsin activity from 34% to 58% of the free trypsin control. Inall cases, Kcat was typically in the range of 1.2–2.5 s-1.

Synthesis and Characterization of Dextrin-MSH. Severalbatches of dextrin-MSH conjugate were synthesized, and after

FPLC fractionation (Figure 8), GPC analysis confirmed conju-gate formation and absence of free MSH (also confirmed bySDS-PAGE, data not shown). The total MSH content was ∼14wt % in pilot scale batches, but after scale-up of conjugateproduction for the biological assays the conjugate contained 37wt % MSH loading.

Stimulation of Melanin Production by Dextrin-MSH((r-Amylase). The melanin production assay was also opti-mized according to the B16F10 cell seeding density, the MSHconcentration used (Figure 9a), and by characterizing the MSHstability and conjugate stability in tissue culture media (Figure9b). As FBS contains amylase, and indeed the dextrin-MSHconjugates were found to degrade in the presence of 10% FBS(Figure 9b), all melanin induction experiments were conductedin heat inactivated 10% FBS. These conditions ((amylase) didnot adversely affect B16F10 viability (results not shown).Melanin production was related to MSH concentration (Figure9a). It was shown that the dextrin-MSH conjugate (37 wt %MSH) stimulated melanin production to a value of 11% the freeMSH control when analyzed using the optimized melanin assayin RPMI + 10% heat-inactivated FBS (Figure 9c). Followingincubation of the dextrin-MSH conjugate with R-amylase (0.2mg/mL), melanin production was increased to 33%. Significantmasking and unmasking of activity was seen (p < 0.05,ANOVA and Bonferroni post hoc test).

Discussion

The two models explored here demonstrated the feasibilityof PUMPT using dextrin for protein/peptide masking, withunmasking possible by addition of R-amylase. As R-amylase

Figure 4. Circular dichroism spectra of dextrin, succinoylated dextrin,free and conjugated trypsin, and succinoylated dextrin plus trypsinat 4 °C in Tris buffer (pH 8.2). Samples were prepared usingsuccinoylated dextrin (47200 g/mol; 26 mol %).

Figure 5. Degradation of dextrin and succinoylated dextrins byR-amylase.

Figure 6. Trypsin activity before and after dextrin conjugation: Effectof the degree of succinoylation.


is present in serum and extracellular fluids, the PUMPT concepthas physiological relevance. Succinoylation was chosen fordextrin functionalization as this chemistry should yield anontoxic intermediate, the resultant linker is relatively stable,16,25

and dextrin succinoylation had been previously optimized.16 Notsurprisingly, it was easier to regenerate enzyme activity byaddition of R-amylase than MSH-receptor binding owing to theeasier accessibility of the low molecular weight substrate to theenzyme’s active site. As shown previously for both chemicallymodified dextrans26 and for dextrin,17 the degree of modificationgoverned the rate of degradation. The extent of regeneration ofprotein/peptide activity in these in vitro models is complex tointerpret. It will be related to (i) the rate of polymer degradation,(ii) liberation of active protein with minimal or no glucoseoligomers attached, and (iii) maintenance of protein or peptideintegrity during the conjugation and regeneration process. Aparticular challenge for design of the in vitro experimentsdesigned to evaluate PUMPT is the difficulty of mimicking theoptimal physiological conditions for polymer degradation while

simultaneously presenting the most favorable environment foractivity of liberated enzyme or ligand–receptor binding.

Dextrin-Trypsin Model. The experiments designed toevaluate R-amylase activation of the dextrin-trypsin conjugatesused a 16 h R-amylase/conjugate incubation period prior tomeasurement of trypsin activity and assay conditions describedpreviously for the comparison of ovine and porcine trypsin usingL-BAPNA.27 Although some immediate regeneration of trypsinactivity has been seen on addition of R-amylase to specificdextrin-trypsin conjugates (results not shown), an exposure timeof 16 h was used here to allow better comparison of the libraryof conjugates, some of which had such a high degree ofsuccinoylation that the polymer degradation was expected tobe relatively slow. Not surprisingly, the highest degree of trypsinmasking was achieved using the higher molecular weight dextrindue to the greater steric hindrance caused by the larger bulkierpolymer chain. Increasing dextrin succinoylation led to greatermasking. Although masking was achieved using the lowermolecular weight dextrin, it was less efficient and there was noobvious relationship in masking between degree of succinoy-lation and conjugate activity. This was not surprising as theaverage weight percent of trypsin in conjugates (thus numberof trypsin molecules per dextrin chain) did not vary greatlybetween samples. Interestingly the Kcat values estimated for allconjugates were comparable to that seen for free trypsin bothhere and for porcine trypsin determined under almost identicalconditions using L-BAPNA (Kcat ) 2.89 s-1).27 Importantly,as far as we are aware, this is the first time that DOSY NMRspectroscopy was used as an analytical technique to definepolymer-protein conjugate composition. Using NMR it waspossible to differentiate between covalent binding and simplepolymer-protein complexation. Measuring diffusion usingDOSY NMR also opens up the possibility of analyzing complexpolymer-protein mixtures and reaction kinetics.

Dextrin-MSH Conjugates. Dextrin-MSH conjugates pre-pared using FPLC fractionation did not contain free MSH. Theestablishment of controlled conditions to enable assay of activityin vitro was, however, complicated. Both succinoylated dextrinand the dextrin-MSH conjugates were degraded over time whenincubated in RPMI + 10% FBS, and although use of heat-inactivated FBS reduced degradation, it did still occur (Figure9b). This was not surprising as MSH degradation by serumenzymes is well-known (reviewed in ref 28). As the in vitroassay measured MSH induction of melanin production byB16F10 cells after 72 h, it was important to establish thataddition of R-amylase would not itself affect cell viability ormelanin production. When incubated in RPMI containing heat-

Figure 7. Reinstatement of trypsin activity following incubation ofdextrin-trypsin conjugates in the presence of R-amylase.

Figure 8. FPLC purification of dextrin-MSH.


inactivated FBS (10%), the activity of the masked conjugatewas reduced to 11%. Addition of R-amylase elevated melaninto 33% the control value. The relatively poor reinstatement of

activity compared to the dextrin-trypsin conjugates might bedue to MSH degradation during the 72 h incubation and/orirreversible inactivation of the MSH by dextrin conjugation. Onepotential site for conjugation is the MSH lysine separated fromthe active site by only one amino acid. Any remaining glucoseoligomers could potentially cause significant steric hindranceof receptor binding.

It should be noted that the extent of regeneration of activityof both conjugate models is only an indication of the potentialextent and rate of unmasking in an in vivo setting. In theseexperiments only one exposure time was used in each model(16 or 72 h) to enable comparisons across a library ofcompounds to be made.

PUMPT, Opportunities and Challenges. PUMPT providesa new strategy with potential to improve delivery of proteinshaving such high inherent toxicity that safe parenteral admin-istration is precluded, to enable the protection of proteinsnormally inactivated while traveling to their therapeutic targetand/or to allow controlled release with time of active proteinsat the target site. PUMPT should be widely applicable to proteinand peptide therapeutics having various kinds of pharmacologi-cal targets, e.g., enzymes, cytokines, growth factors, andantibodies. Our recent studies have successfully applied thedextrin-R-amylase strategy for the delivery of epidermal growthfactor for use in the context of wound repair29 and forphospholipase A2 as an anticancer agent.30 Moreover, experi-ments with hyaluronic acid and hyaluronidase31 have verifiedthe suitability of other polymers and activation strategies forPUMPT.

For further PUMPT development it will be essential tooptimize the conjugation chemistry on a case-by-case basisdepending on the protein therapeutic to be modified, to minimizepolymer polydispersity thus minimizing conjugate heterogeneity,and improve the purification techniques to ensure removal offree polymer. The trypsin and MSH conjugates described herewere pure in respect of free protein, but DOSY NMR suggestedpresence of free dextrin in the dextrin-trypsin conjugates. Inaddition, the identity and biocompatibility of the primarymetabolites of PUMPT must be characterized to ensure safeelimination.

Acknowledgment. H.G. thanks BBSRC for supporting herPhD studentship and M.J.V. is grateful for a Marie CurieFellowship, Contract No. HPMF-CT-2002-01555, for support.R.J.C. and M.J.V. are currently researchers from the Ramón yCajal program (MEC, Spain), and they would also like to thankDr. A. Pineda-Lucena for helpful discussions.

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Figure 9. Experiments designed to investigate the biological activityof MSH (control) and dextrin-MSH conjugates in B16F10 cells. (a)Control studies designed to define the effect of MSH concentrationon melanin production (72 h) in B16F10 cells (seeded to a density of2.5 × 104 cells/mL), and data represent the mean (n ) 6) ( standarddeviation. (b) Effect of media composition on the degradation (byGPC) of dextrin (47200 g/mol), succinoylated dextrin (26 mol %), anddextrin-MSH when incubated for 16 h at 37 °C. (c) Melaninproduction (72 h) in B16F10 cells incubated with MSH or dextrin-MSH((amylase 0.2 mg/mL) (10-5 M MSH equiv). Cells were incubatedin RPMI + 10% heat-inactivated FBS at a seeding density of 2.5 ×104 cells/mL. Data shown represents the mean (n ) 12–18) ( SEM.Statistical significance (/) indicates p < 0.05 measured using ANOVAand Bonferroni post hoc test and comparing each treatment and thecontrol.


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