improved tumor-speciﬁc drug accumulation by polymer ...improved tumor-speciﬁc drug accumulation...

Large Molecule Therapeutics

Improved Tumor-Specific Drug Accumulation byPolymer Therapeutics with pH-Sensitive DrugRelease Overcomes Chemotherapy ResistanceAnne-Kathrin Heinrich1, Henrike Lucas1,2, Lucie Schindler3, Petr Chytil3, Tom�a�s Etrych3,Karsten M€ader1, and Thomas Mueller2

Abstract

The success of chemotherapy is limited by poor selectivityof active drugs combined with occurrence of tumor resistance.New star-like structured N-(2-hydroxypropyl) methacrylamide(HPMA) copolymer-based drug delivery systems containingdoxorubicin attached via a pH-sensitive hydrazone bond weredesigned and investigated for their ability to overcome chemo-therapy resistance. These conjugates combine two strategiesto achieve a high drug concentration selectively at the tumor site:(I) high accumulation by passive tumor targeting based onenhanced permeability and retention effect and (II) pH-sensitivesite-specific drug release due to an acidic tumor microenviron-ment. Mice bearing doxorubicin-resistant xenograft tumorswere treated with doxorubicin, PBS, poly HPMA (pHPMA) pre-cursor or pHPMA–doxorubicin conjugate at different equivalentdoses of 5 mg/kg bodyweight doxorubicin up to a 7-fold totaldose using different treatment schedules. Intratumoral drug

accumulation was analyzed by fluorescence imaging utilizingintrinsic fluorescence of doxorubicin. Free doxorubicin inducedsignificant toxicity but hardly any tumor-inhibiting effects.Administering at least a 3-fold dose of pHPMA–doxorubicinconjugate was necessary to induce a transient response, whereasdoses of about 5- to 6-fold induced strong regressions. Tumorscompletely disappeared in some cases. The onset of responsewas differential delayed depending on the tumor model, whichcould be ascribed to distinct characteristics of the microenvi-ronment. Further fluorescence imaging–based analyses regard-ing underlying mechanisms of the delayed response revealed arelated switch to a more supporting intratumoral microenvi-ronment for effective drug release. In conclusion, the currentstudy demonstrates that the concept of tumor site-restrictedhigh-dose chemotherapy is able to overcome therapy resistance.Mol Cancer Ther; 15(5); 998–1007. �2016 AACR.

IntroductionAs cancer is a leading cause of death worldwide, there is a high

effort to find new therapies. Although there is already amultitudeof antineoplastic agents, the success of chemotherapy is oftenlimited. Major obstacles in cancer therapy are dose-limitingsystemic toxicities, due to nonspecific drug delivery and chemo-therapy resistance (1–4). Achieving higher intratumoral drugconcentrations while protecting normal tissue would improvetherapy outcome and diminish severe side effects. The increasingimportance of resistant tumors is a huge challengewhich has to betackled.

Since many years, the enhanced permeability and retention(EPR) effect, discovered by Matsumara and Maeda (5, 6), is well

known to enable a passive tumor targeting of macromolecules. Avariety of nanoscaled drug delivery systems taking advantage ofthe EPR effect were developed until now, for example, nanopar-ticles, liposomes, polymeric micelles, dendrimers, or polymer–drug conjugates (7). Because of the target-oriented drug delivery,they are promising concepts to optimize tumor therapies and toovercome resistance (4, 8, 9). Furthermore, a site-specific drugrelease can result in higher drug concentrations exclusively intumor tissue. It can be triggered by different stimuli like temper-ature, redox status, pH, or enzymes (8–10). Tumors are known tohave a slightly acidic microenvironment compared with normaltissues (11–13). A pH gradient between intracellular and extra-cellular compartments arises among other contributing factorsfrom the high glycolysis rates of cancer cells (7). The occurrence ofhypoxia within tumor tissue also supports the acidification of themicroenvironment due to the hypoxia-induced cascade whichleads to anupregulation of carbonic anhydrase IX (14). Because ofthe different values in healthy and tumor tissue, pH is a promisingstimulus to achieve a higher drug release selectively at the tumorsite.

Referring to this, we recently investigated new polymeric drugdelivery systems (pDDS) based on N-(2-hydroxypropyl) metha-crylamide copolymers (pHPMA) which contain a fluorescent dyeacting as a model drug coupled via pH-dependent hydrolyticallycleavable hydrazone bond (15, 16). This bond is rather stableunder physiologic conditions (pH 7.4) and cleavable in slightacidicmilieu (16). That enables a drug release preferentially in thetumor whereas the toxic side effects are decreased because of

1Institute of Pharmacy, Martin Luther University Halle-Wittenberg,Halle (Saale), Germany. 2Department of Internal Medicine IV, Oncol-ogy/Hematology, Martin Luther University Halle-Wittenberg, Halle(Saale), Germany. 3Institute of Macromolecular Chemistry AS CR,Prague, Czech Republic.

Note: Supplementary data for this article are available at Molecular CancerTherapeutics Online (http://mct.aacrjournals.org/).

Corresponding Author: Thomas Mueller, Department of Internal Medicine IV,Oncology/Hematology, Martin Luther University Halle-Wittenberg, Ernst-Grube-Str. 40, 06120Halle (Saale), Germany. Phone: 49-345-557-7211; Fax: 49-345-557-7279; E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-15-0824

�2016 American Association for Cancer Research.

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negligible peripheral release. Furthermore, the pDDS were stablylabeled with another fluorescent dye to follow their in vivodistribution. Two types of pHPMA-based pDDS differing inpolymeric chain structure were investigated: a linear structuredwith a molecular weight of 30 kDa and a new, star-like structuredwith 200 kDamolecular weight. Using noninvasive multispectralfluorescence imaging (msFI), wewere able to detect the pDDS andthe model drug signal simultaneously and studied the in vivo fateof those conjugates and release of model drug in living mice. Theevaluation of the imaging data indicated a high tumor-specificaccumulation ofmodel drug confirming the concept and revealedimproved characteristics of the star-like structured pDDS (15). It iswell known that pHPMAconjugateswithhighermolecularweightenable improved tumor accumulation due to prolonged circula-tion. Until now, linear structured systems, for example, multi-block copolymers were investigated preferentially. As the star-likestructure showed superior tumor accumulation in our prelimi-nary studies (15), we decided to determine the antitumor efficacyof these conjugates. For the current study, the model drug wasreplaced by doxorubicin as a real chemotherapeutic drug andtherapeutic efficiency of this new star-like structured pHPMA–doxorubicin conjugate was investigated for the first time indoxorubicin-resistant tumor models. Doxorubicin was selectedfor its importance as an anticancer agent. In addition, its intrinsicfluorescence can permit the analyses of the intratumoral drugaccumulation using FI. The aim of the study was to analyzewhether a sufficient high intratumoral drug dose accumulationcan be achieved by this new star-like shaped pHPMA–doxorubi-cin conjugate with pH-sensitive drug release to induce an effectiveresponse in drug-resistant tumors.

Materials and MethodsMaterials

Doxorubicin hydrochloride (Dox-HCl), PBS, RPMI1640medi-um, trypsin, and sulforhodamine B (SRB) were purchased fromSigma-Aldrich Chemie GmbH. Trichloroacetic acid (TCA) andTris(hydroxymethyl)-aminomethane (TRIS) were obtained fromCarl Roth GmbH & Co. FBS was purchased from Biochrom AG.Streptomycin/penicillin was purchased from Merck ChemicalsGmbH. Furthermore, the fluorescent dyes DY-782 and DY-676were obtained from Dyomics GmbH. The FI agent Hypoxisensewas purchased from PerkinElmer. Isoflurane for veterinary use(Forane) was obtained from Abbott.

Polymer–drug conjugatesThe synthesis of polymer–drug conjugates and the detailed

physicochemical characterization is presented in the Supplemen-tary section. Two high molecular weight star-like structured con-jugates were used for the experiments. pHPMA conjugate 1(pHPMA–doxorubicin conjugate) was loaded with approximate-ly 10%wt doxorubicin linked via pH-dependent cleavable hydra-zone bond. pHPMA conjugate 2 was also loaded with approxi-mately 10% wt doxorubicin linked via hydrazone bond andadditionally 0.6% wt near-infrared (NIR) fluorescent dye (DY-782, Dyomics GmbH). The content of fluorescent dye and doxo-rubicin in polymer conjugates (Supplementary Table S1) wasdetermined by spectrophotometry on a Helios a spectrophotom-eter (Termochrom). For calculation, molar absorbation coeffi-cients of the DY-782 (eDY-782¼ 170,000 L/mol//cm in ethanol)and of the doxorubicin (eDox ¼ 11,200 L/mol/cm in methanol)

were used. The accuracy of determination of the doxorubicinamount byUV-VIS spectroscopy was validated in previous studiesby another method based on selective hydrolysis of glycosidebond in doxorubicin attached onto the polymer prodrug andconsequent HPLC determination of formed aglycon of doxoru-bicin (17). Both methods gave the same results with variation upto 4%.

Cell lines and in vitro testsCell lines A2780 and A2780cis were provided by Prof. G.

Bendas (Pharmaceutical Institute, Rheinische Friedrich-Wil-helms-University Bonn, Germany) in 2014. Originally, both celllines were purchased from the European Collection of Authen-ticatedCell Cultures (ECACC). Theywere reauthenticated in 2015by the Leipniz Institute DSMZ-GermanCollection ofMicroorgan-isms andCellCultures using short tandemrepeat analysis. The cellline 1411HP was provided by Prof. P. W. Andrews (Centre forStemCell Biology, University of Sheffield, Sheffield, UK) in 1997.Since then, the cells were uninterruptedly controlled by the sameperson (T. Mueller). The identity of the cells is proved by gener-ating xenograft tumors followed by histologic examination. The1411HP tumors are composed of embryonal carcinoma withanaplastic or epithelial appearance and differentiated structuresof yolk sac tumor as described and shown in Fig. 3 in the originalpublication of Vogelzang and colleagues (1985). No additionalauthentication of the cell lines was performed. All cell lines werecultivated with RPMI medium (containing 10% FBS and 10%penicillin/streptomycin).

For cytotoxicity assays, cells were seeded in 96-well plates for24 hours and incubated at 37�C/5% CO2. After 24-hour serialdilutions of doxorubicin-stock solution (1.44 mmol/L) wereprepared (0.001 mmol/L–10 mmol/L) and added to the cells.Furthermore, the pHPMA–doxorubicin conjugates were prein-cubated for 24 hours in phosphate buffers with different pHvalues ranging from 5.5 to 7.4. Afterward, they were useddirectly to prepare the serial dilutions similar to free doxoru-bicin. Then cells were incubated for 2 hours. Afterward, thesupernatant was removed and fresh RPMI medium was addedto the cells for another incubation period of 96 hours. Finally,the supernatant was removed and cells were fixed by adding10% (w/v) TCA. Well plates were stored at 4�C for at least 2hours. After some washing steps with distilled water, cells werestained with SRB [0.4% in 1% (v/v) acetic acid]. 100 mL of theSRB solution were added to each well. Plates were left at roomtemperature for 30 minutes and then rinsed with 1% (v/v)acetic acid to remove the unbound dye. Afterward, plates wereallowed to dry at room temperature. For the measurements,100 mL of 10 mmol/L Tris buffer were added to each well andplates were read at 570 nm using a microplate reader.

For the determination of the IC50 and IC90 values, dose–response curves were evaluated using Excel software showing theconcentration-dependent cell growth inhibition (%). Exact IC50

and IC90 values were then calculated by linear interpolation.

Animal care and treatmentMale athymic nude mice (Hsd:Athymic Nude-Foxn1nu, male;

fromHarlanWinkelmann)were kept under controlled conditions(12-hour day/night cycle, 24�C). After 2weeks of setting inperiod,they were short-time anesthetized using isoflurane for tumor cellinjection. Tumor cells (either 1�107 cells 1411HP, 5�106 cells

Polymer Therapeutics Overcome Chemotherapy Resistance

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A2780or 5�106 cells A2780cis), suspended in 150mL of PBSweresubcutaneously injected to the right side of the mice. Mouseweight and tumor sizeweremeasured continuously. Furthermore,tumor volume (V)was estimated after calipermeasurement based

on length (l) andwidth (w) by using the equation:V¼ p/6� l�w2

according to Tomayko and colleagues (18). The increase of thetumor volume normalized to day 0 or first day of treatments (d1)was then displayed over time.

Figure 1.Structure of pHPMA–doxorubicin (Dox) conjugates.A, scheme of the star-like structure of the pHPMA–doxorubicin conjugates. pHPMAconjugate 2 contains acovalently linked fluorescent label DY-782 (-X) and acleavable, pH-sensitive linked drug doxorubicin (¼Y).pHPMA conjugate 1 instead contains only the pH-sensitive linked doxorubicin. B, detailed molecularstructure of the pHPMAside chains that are attached tothe PAMAM [Poly(amidoamine)] dendrimer core ofthe star-like conjugates. Arrows point to bonding siteof either doxorubicin or DY-782.

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To compare the in vivo doxorubicin sensitivity of the resistantmodels 1411HP and A2780cis with the sensitive ovarian carci-noma model A2780, respectively, 10 1411HP, A2780cis, andA2780 tumor xenograft bearing athymic nude mice were evensubdivided into PBS control groups (each n¼ 5) and doxorubicintherapy groups (each n¼ 5).When the tumors reached an averagesize of 0.3 cm3 � 0.008 cm3 in each group, treatments werestarted. Mice received either PBS or doxorubicin (5 mg/kg BW)intravenously at days 1, 4, and 9 (A2780 at day 10 instead).

To investigate therapeutic efficacy of the pHPMA-conjugate 1(pHPMA–doxorubicin conjugate), 29 1411HP-tumor xenograftbearing mice were divided into four groups according to theirtumor size (mean tumor volume 0.25� 0.02 cm3) and got eitherinjections of pHPMA–doxorubicin conjugate, free doxorubicin,pHPMA precusor or PBS. The pHPMA–doxorubicin conjugateand also pHPMA precursor were dissolved in PBS (according to aDoxorubicin concentration of 1.25 mg/mL). All injection solu-tions were sterile filtered before injection (0.2 mm Millex) andinjected into the tail vein of mice 15 days after tumor cellinoculation.

Furthermore, the doxorubicin accumulation in tumors wasexamined. Therefore, 10 1411HP tumor xenograft bearing athy-mic nudemice were treated with either free doxorubicin (5mg/kgBW), 1-, 2- and 3-fold doxorubicin equivalent dose of thepHPMA–doxorubicin conjugate or PBS as a control when tumors

had an appropriate volume for imaging procedure (0.75 � 0.25cm3). 48 hours after intravenous injection, mice were sacrificed,tumors necropsied, and examined by msFI to compare the doxo-rubicin accumulation.

To determine the effect of different treatment schedules, 91411HP and 3 A2780cis tumor xenograft bearing athymic nudemice were treated with either pHPMA–doxorubicin conjugate orPBS.

For examinations of the tumor micromilieu, again 1411HP-tumor xenografts were established in 6 athymic nudemice. Whentumor volume was appropriate (0.75 � 0.25 cm3), differenttreatment schedules were tested. The schedules comprised injec-tions of either a combination of pHPMA–doxorubicin conjugateswith following Hypoxisense (Perkin Elmer) injection (100 mL inPBS; 2 nmol/100 mL) or only the Hypoxisense injection as acontrol. 24 hours after Hypoxisense injection, mice were sacri-ficed, tumors were necropsied and cross-sectioned to performex vivo msFI.

All experiments complied with regional guidelines and regula-tions and were approved by the local authority in Saxony-Anhalt.

In vivo and ex vivo msFINoninvasive msFI was carried out on a Maestro in vivo imaging

system from CRI (Cambridge Research and Instrumentation).Image Cubes were acquired in the range of 500 to 720 nm in

Figure 2.Analyses of tumor response totreatmentwith doxorubicin (Dox) andpHPMA–doxorubicin conjugate. A,tumor volume (TV) increasenormalized to day 0 over time. PBScontrol groups (means� SD; each n¼5) and doxorubicin therapy groups(means � SD; each n ¼ 5). Micereceived either PBS or doxorubicin(5 mg/kg BW, i.v.) at day 1, 4, and 9(A2780 at day 10 instead). In eachtherapy group, one mouse needed tobe sacrificed because of severetoxicity already at day 9 (1411HP) andday 10 (A2780). Arrows point totreatments. B, TV increase normalizedto day 0 over time of 1411HP tumor-bearing mice. PBS control group(means � SD; n ¼ 3) and pHPMAprecusor, doxorubicin and pHPMA–doxorubicin conjugate group (means� SD; each n¼6). DeterminedP valueon day 8 (all groups underconsideration) was 0.107 (Kruskal–Wallis test) indicating no significantdifference. On day 12, P ¼ 0.639 wasdetermined (Student t test) indicatingno statistical difference at this timepoint. Monitoring was completedwhen at least 2 mice within a grouphad reached maximal tolerated TV. Inthe doxorubicin group, one mouseneeded to be sacrificed already at day12 whereas the remaining mice werefolloweduntil day 16whennext 3micehad reached maximal tolerated TV.Arrows point to treatments.






10nmstepsusing thebluefilter set (Excitationfilter, 445–490nm;emission filter, 515 nm long-pass) to detect the doxorubicinsignal. The NIR filter set (excitation filter, 710–760 nm; emissionfilter, 800 nm long-pass) was used to acquire cubes in the range of780 to 950 nm for detecting the polymer (DY-782) signal.Furthermore, the red filter set (excitation filter, 615–665 nm;emission filter, 700 nm long-pass; cube acquisition from 680 to950 nm, 10 nm steps) was used for detection of the Hypoxisensesignal. The images were automatically exposed to avoid overex-posure or underexposure. To separate the single spectral species ofdoxorubicin, DY-782, and Hypoxisense from background andautofluorescence spectra, Maestro software (version: 2.10.0.) wasused. The intensity weighted images were displayed using the hotcolor profile. During imaging process, mice were anesthetizedwith isoflurane (Forane, Abbott) for veterinary use in oxygen (2 L/minute) using an initial dose of 2.5% and a maintenance dose of1%–2%. Furthermore, mice were placed at a warming plate(35�C)during anesthesia to avoid adecrease of body temperature.

In addition, the necropsied tumorswere examined. Therefore, acertain time after respective injection mice were sacrificed andtumorswere necropsied, cross–sectioned, and examinedusing theblue, red, orNIRfilter set depending on the previous injection. Forthe comparison, the measured fluorescence intensities were nor-malized by the tumor area and exposure time.

For microscopic examinations, the necropsied tumors werecross-sectioned, fixed in 4% formalin, embedded in paraffin,sliced (3 mm), dewaxed, and stained with hematoxylin & eosin(H&E) to be observed by light microscopy.

ResultsTwo variants of the 200 kDa star-like structured pHPMA-based

polymer conjugates with doxorubicin attached via a pH-sensitivehydrazone bond were synthesized (see supplemental section forsynthesis and more detailed physicochemical characterization).Doxorubicin was chosen due to its excellent ability to be linked tothe polymer backbone via hydrazone bond and furthermorebecause of its intrinsic fluorescence which was required for the

FI analyses. The pHPMA conjugate 1 was applied to prove ther-apeutic efficacy. Moreover, a second pHPMA conjugate was syn-thesized containing, in addition, a covalently linked NIR fluores-cent label (DY-782) enabling the determination of the in vivo fateof the polymer conjugate (pHPMA conjugate 2; Fig. 1). In addi-tion, a pHPMA precursor (contains no doxorubicin) was used ascontrol treatment.

An important prerequisite for proving the potential of thepHPMA conjugates to overcome chemotherapy resistance was toemploy a doxorubicin -resistant tumor model. Therefore, in vitrocytotoxicity testing with doxorubicin in a panel of tumor cell linesfrom different entities was performed. Among them only one, theA2780ovarian carcinoma cell line, showed anapparent sensitivityto doxorubicin with an IC50 of 3 nmol/L. All other cell lines haddistinctly higher IC50 ranging from 15 to 35 nmol/L. Subsequent-ly, it was tested whether the differences in in vitro sensitivity todoxorubicin can be reproduced in vivo in derived xenografttumors. As potential doxorubicin-resistant tumor models themultidrug resistant germ cell tumor cell line 1411HP (IC50

doxorubicin: 35 nmol/L) and the cell line A2780cis (IC50 doxo-rubicin: 17 nmol/L) which is also cross-resistant to doxorubicinwere selected. The cell line 1411HP represents a model of naturaldrug resistance as these cells were derived from a tumor of apatient with refractory disease after chemotherapy (19, 20),whereas the acquired drug resistance of A2780cis was artificiallyestablished derived from the cell line A2780 (21). In addition, thederived xenograft tumors of both cell lines typically show verysimilar characteristics. They are characterized by a loose structuredtumor tissue exhibiting a lot of vital cells and a lack of necrotic/fibrotic areas at least in smaller tumors. Furthermore, they exhibitonly a low amount of extracellular matrix are highly vascularizedand showhigh growth rates. As shown in Fig. 2A, treatment with 5mg/kg bodyweight doxorubicin on day 1 and day 4 inducedregressions in sensitive A2780 tumors, which however turnedinto regrowth within few days. Because of occurrence of treat-ment-related toxic side effects (loss of bodyweight), the nextinjection was possible on day 9. The third doxorubicin applica-tion induced no further tumor regression and resulted in a

Figure 3.Impact of treatment schedule and total dose on tumor response. Plotted tumor volume (1411HP tumors) increase normalized to first day of treatment (d1) over time.Different doses (A, 3- to 5-fold overall doxorubicin equivalent dose; B, 6-fold overall doxorubicin equivalent dose) of pHPMA–doxorubicin conjugate wereadministered in different time intervals (see legend). At least an overall dose of 5- to 6-fold doxorubicin equivalent dose of the pHPMA–doxorubicin conjugate wasnecessary to be administered within 10 days for achieving a tumor regression.

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pronounced toxicity (loss of bodyweight, sometimes accompa-nied by atypical behavior) in some cases allowing no furthertreatment. Nevertheless, doxorubicin treatment resulted in a cleartumor growth inhibition compared with control tumors in sen-sitive A2780 tumors and a further control of tumor growth bytreatment with doxorubicin seemed possible at least in miceshowing a higher tolerance of doxorubicin. In resistant 1411HPtumors, the same treatment schedule induced only little growthretardations (Fig. 2A) but also similar treatment-related toxiceffects with severe toxicity (loss of more than 20% of initialbodyweight) in one mouse after the third injection of doxorubi-cin. Furthermore, similar negligible growth retardation effects inresponse to doxorubicin treatment were observed in A2780cistumors (Supplementary Fig. S1). In summary, these analysesdemonstrated that doxorubicin treatment with dosages aroundthe MTD resulted in a clear but not prolonged response of asupposed doxorubicin-sensitive tumor in the used nude mousemodel. Notably, we observed a very different extend of doxoru-bicin-related toxicity among the mice ranging from good toler-ability to severe side effects. The clear in vitro doxorubicin resis-tance of 1411HP and A2780cis was confirmed in vivo.

First, the stably fluorescence-labeled pHPMA–doxorubicinconjugate (conjugate 2) was used to prove tumor accumulationin both models in vivo. FI analyses revealed enhanced tumor-specific accumulation in accordance with our previous analyseswith the pHPMA polymer conjugates that contained a fluorescentmodel drug instead of doxorubicin (data not shown). Thentherapeutic efficacy was tested and pHPMA–doxorubicin conju-gate 1 was compared with free doxorubicin, unloaded pHPMA-precursor and PBS in the resistant 1411HPmodel using the sametreatment schedule as above. Treatment with doxorubicininduced increasing signs of toxicity but hardly any tumor inhibit-

ing effects as expected frompreliminary trials (Fig. 2B). Treatmentwith pHPMA precursor had no impact on tumor growth and wasshown to be completely nontoxic (Fig. 2B). The pHPMA–doxo-rubicin conjugate was given once at equivalent dose of 5 mg/kgbodyweight doxorubicin and no obvious tumor response wasobserved. As the drug carrier was expected to release the drugpredominantly in the tumor avoiding systemic toxicity, it wasdecided to administer the 3-fold dose equivalent of 15 mg/kgdoxorubicin on day 4 and 9. The changed treatment scheduleinduced some signs of toxicity associated with weight loss (Sup-plementary Fig. S2) but no clear tumor response could be noticed.WeperformedKruskal–Wallis test onday8which led toP¼0.107indicating that there is no statistical significant difference betweenthe groups. Unexpectedly, after the third injection (day 9), thetumors started to continuously regress reaching a nonmeasurablestage for 80 days and even completely disappeared in 3mice (Fig.2B). The only reasonable time point where statistical significantdifference could have been tested was on day 12 [P ¼ 0.639 (nosignificant difference), Student t test]. Afterward, onemouse of thefree doxorubicin group needed to be sacrificed because of tumorburden and toxic side effects (weight loss). Moreover, already atday 16, the next 3 mice needed to be sacrificed. For correctstatistical analyses, all mice of each group should be taken underconsideration. As this was not possible after day 12, no P valuecould be determined proving significant differences between freedoxorubicin and pHPMA–doxorubicin conjugate group. Never-theless, clear superiority of the pHPMA–doxorubicin conjugatetreatment could be visualized additionally by Kaplan–Meier plot(Supplementary Fig. S3). During tumor regression, mice of thepHPMA–doxorubicin conjugate group regained the lost weight.

In a current experiment with A2780cis tumors, the sametreatment schedule also induced tumor regression but the onset

Figure 4.Evaluation of intratumoraldoxorubicin accumulation. A,fluorescence images of necropsiedand cross-sectioned 1411HP tumors 48hours after injection of either freedoxorubicin (5 mg/kg bodyweight),1-, 2-, and 3-fold doxorubicinequivalent dose of the pHPMA–doxorubicin conjugate or PBS as acontrol. Dark red indicates lowintensity whereas yellow/whiteindicates high fluorescence intensity(doxorubicin signal). Increasing doseled to increasing doxorubicinaccumulation. B, bar plot of measuredmean fluorescence intensities (FI) ofnecropsied and cross-sectioned1411HP tumors (doxorubicin signal) of2 trials (each group, n ¼ 2). FIs werenormalized by the tumor area andexposure time [average signal (scaledcounts/s)]. Plot contains standarddeviations (small black bars).






of response occurred earlier compared with 1411HP tumors andhigher treatment-related toxicity was observed.

Next it was assessed, whether a specific dose, that has to beadministered to induce tumor regression, can be determined.Furthermore, it was of interest whether the characteristic patternof tumor response observed in the initial trial might be a conse-quence of the specific treatment schedule. Therefore, differenttreatment schedules were tested to disclose the impact of possiblekey parameters as number, interval, and single dose of injectionsas well as the total dose within the therapy schedule. An appli-cation of three 2-fold doses on days 1, 4, and 9 resulted in thecharacteristic pattern of delayed but strong response as observedin the initial trial (Fig. 3B). Interestingly, treatment with 2-folddoses using the narrower interval ondays 1, 3, and5 resulted innoobvious differences in the pattern of response. Tumor regressionalso started after day 10 (Fig. 3B). However, this schedule inducedmore toxic effects. To proof a possible necessity of a third injectionfor tumor response, mice were treated only two times with 3-fold

doses on days 1 and 4 avoiding third injection while adminis-tering the same total drug dose. Again, the process of clear tumorregression was observed after day 10 independent of a thirdinjection on day 9 (Fig. 3B). Furthermore, the specific patterncould also be observed using a larger interval with injection of two3-fold doses on days 1 and 8 (Fig. 3B). Besides, this weeklytreatment scheme seemed to be better tolerated. In a second setof treatments, the total dose was reduced. Application of anoverall 5-fold dose, given as either 2 or 5 single injections, inducedthe characteristic pattern of response but total doses below that (3-and 4-fold total dose) resulted in an incomplete tumor responsewith early tumor regrowth after a first reaction (Fig. 3A). Thesedata suggest that the characteristic response of the resistant1411HP tumors is not dependent on dose interval but a sufficienthigh total dose (5- to 6-fold total dose) has to be administeredwithin 10 days to induce a complete response.

Similar to 1411HP tumors, application of a 5-fold total dosewas sufficient to induce an effective response in A2780cis tumors

Figure 5.Analyses of time and treatment-related switch of tumor microenvironment. A, fluorescence images of necropsied and cross-sectioned 1411HP tumors. Micereceived either only Hypoxisense as a control (upper sample) or 3-fold doxorubicin (Dox) equivalent dose of pHPMA–doxorubicin conjugate 1 once (nonrespondingtumor, middle sample) or twice (d1 and d4, responding tumor, lower sample) with following Hypoxisense injection. Dark red indicates low intensity whereasyellow/white indicates high fluorescence intensity (FI; Hypoxisense signal). Clear accumulation of Hypoxisense can only be detected in the tumor thatshowed a response. B, bar plot of mean FIs (Hypoxisense signal) of samples (1411HP tumors) shown in A and C (bottom) after intravenous administration ofpHPMA conjugate 1 and pHPMA conjugate 2, respectively, with following Hypoxisense injection. FIs were normalized by tumor area and exposure time. Plotcontains standard deviations (small black bars). C, composite image (top) of single signals (bottom). Single signalswere allocated certain colors, signal overlay led tomixed colors. A clear increased intratumoral content of pHPMA conjugate 2 after treatment with two 3-fold doses was confirmed which was indicated byboth, the doxorubicin- and the polymer-derived signal. An increased intensity of Hypoxisense signal was observable in responding 1411HP tumor. A purepolymer-derived signal was detectable at the rim of the responding 1411HP tumor indicating pHPMA conjugate 2 after release of doxorubicin.

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too, whereas 3- and 4-fold doses resulted in an incomplete tu-mor response (Supplementary Fig. S1). However, even treatmentwith a single 3-fold dose had a clear improved impact to inhibittumor growth compared with free doxorubicin treatment. Again,the onset of response of A2780cis tumors was observed to beearlier as compared with 1411HP tumors.

To prove an expected higher doxorubicin accumulation intumors after pHPMA–doxorubicin conjugate administration,mice bearing 1411HP tumors (2 per group) were treated witheither free doxorubicin, the 1-fold, 2-fold, or 3-fold doxorubicinequivalent dose of the pHPMA–doxorubicin conjugate and 48hours after intravenous injection, the necropsied and cross-sec-tioned tumors were analyzed by FI. As shown in Fig. 4, even the 1-fold dose led to a clear improvement compared with free doxo-rubicin which directly demonstrates the effect of the pHPMA-carrier. Furthermore, the application of increasing doses ofpHPMA–doxorubicin conjugate resulted in stepwise increaseddoxorubicin-derived signals indicating improved drug delivery(Fig. 4).

Next studies were conducted to investigate the possible under-lyingmechanismof the differential delayed response to treatmentwith pHPMA–doxorubicin conjugate. Initially, the kinetics of pH-dependent drug release and activation were investigated in vitro.The pHPMA–doxorubicin conjugate was preincubated in bufferswith different pH values ranging from 7.4 to 5.5 for 24 hours anddirectly used for cytotoxicity assays in comparison with freedoxorubicin. A gradual lowering of the pH resulted in a stepwiseincrease of cytotoxicity in both cell models. Nearly the completeamount of doxorubicin seemed to be released at pH 5.5, resultingin a comparable dose–response curve and IC50 value as freedoxorubicin (Supplementary Fig. S4). This confirmed the mech-anism of pH-dependent drug release by pHPMA–doxorubicinconjugate and suggested that rather xenograft-specific character-istics are responsible for the different delayed responses. Besides, adifference in cytotoxic behavior comparing pH 7.4 preincubatedand not preincubated pHPMA–doxorubicin conjugate wasnoticed. This indicated the release of a small amount of doxoru-bicin also at a physiologic pH value.

It was hypothesized that the microenvironment of 1411HPtumors initially does not enable an efficient drug release. To provethis, we took advantage of the reproducible and reliable effect thatthe onset of noticeable tumor response occurred around day 10.For the characterization of the tumor microenvironment, the FIagent Hypoxisense (Perkin Elmer) was injected intravenouslyprior to imaging analyses. It is targeted to carbonic anhydrase IX(CA IX) which is known to be overexpressed by cells due to localhypoxia (14, 22). As the catalyzed reaction of CA IX leads to alower pH in surrounding tissue (interstitial compartment) hyp-oxia indicates a supporting condition for the effective drug release

from the pHPMA–doxorubicin conjugate. Tumors were necrop-sied and cross-sectioned formsFI. First, we tested normal growing1411HP tumors, a small and a bigger one. There was nearly noaccumulation of the Hypoxisense agent detectable in the tumor(Fig. 5A, top). Then mice were treated with the pHPMA–doxo-rubicin conjugate (pHPMA conjugate 1) used in therapy studiesand in a second experiment, with the pHPMA conjugate 2 (stablylabeled with the NIR dye) to be able to track polymer accumu-lation independent of the doxorubicin signal. In each experiment,one treatment schedule included an injection of a 3-fold dose onday 1 followed by injection of Hypoxisense on day 4 afterconfirmation of lack of response. Second, schedule comprisedtreatment with 3-fold doses on days 1 and 4 followed applicationof Hypoxisense on day 9 after confirmation of first signs ofresponse. As shown in Fig. 5 only in the responding tumors, aclear accumulation of the agent was noticeable. These analysesclearly showed that an alteration of the tumormicroenvironmenthas occurred at a timewhen the tumor regression process typicallystarts but not much earlier. Furthermore, a clear increased intra-tumoral content of pHPMA conjugate 2 after treatments with two3-fold doses was confirmed which was indicated by both, thedoxorubicin- and the polymer-derived signal (Fig. 5C, bottom).In addition, a composite analysis of single signal was performed.As shown in Fig. 5C (top), signals of doxorubicin and Hypox-isense were closely related andmost prominent in the respondingtumor. A pure polymer-derived signal was observed at the rim oftumors (colored in green) which indicates pHPMA conjugate 2after release of doxorubicin. In addition, analyses of A2780cistumors using Hypoxisense indicated a more hypoxic/acidicmicroenvironment compared with 1411HP tumors (Supplemen-tary Fig. S5). It can be assumed that the conditions in thisxenograft model are more sufficient for the drug release. Further-more, these tumors showed a therapy response already after thefirst injection which corroborates the theory of a more sufficientmicroenvironment.

To get an intention of changes in the tumors under therapyon cellular level, histologic examinations of 1411HP tumorswere made. Formalin-fixed and paraffin-embedded tumor sec-tions were microscopically examined after HE staining. A cleardifference between untreated, still nonresponding andresponding tumors was observed. The overall tissue structureof responding tumors (2�3-fold dose administered) was char-acterized by stressed, more loosely structured, swollen cells(Fig. 6, right) accompanied by many apoptotic areas whereasthe nonresponding tumors (1�3-fold dose administered) rath-er resembled to untreated tumors (Fig. 6, middle and left).These analyses confirmed the observation of a delayed tumorresponse on a histologic level and were in accordance withresults obtained by FI.

Figure 6.Histologic analysis of the treatment-relatedchange of tumor tissue structure. H&E-stainedslices of untreated, still nonresponding andresponding 1411HP tumors after necropsy.Tissue structure of responding tumors ischaracterized by stressed, more looselystructured, swollen cells (right) accompaniedby many apoptotic areas whereas the non-responding 1411HP tumors rather resembledto untreated 1411HP tumors (middle and left).






DiscussionIn the current study, new 200 kDa star-like structured pHPMA-

based polymer conjugates with doxorubicin attached via a pH-sensitive hydrazone bond were synthesized and investigated fortheir ability to overcome chemotherapy resistance. These con-jugates combine two strategies to achieve a high drug concentra-tion selectively at the tumor site: (I) high accumulation of poly-mer drug conjugates by passive tumor targeting based on EPReffect and (II) pH-sensitive site-specific drug release due to anacidic tumor environment. Using two tumor models, one with anatural (1411HP) and the other with acquired resistance(A2780cis), the ability of these conjugates to overcome chemo-therapy resistance could be demonstrated.

Both models were selected on the basis of in vitro cytotoxicitytesting which revealed a clear doxorubicin resistance of both celllines compared with the sensitive cell line A2780, although to adifferent extent. The clear doxorubicin resistance could be repro-duced in vivo and doxorubicin treatment in relevant dosagesinduced hardly any tumor inhibiting effects in both xenograftmodels. Interestingly, the degree of resistance to doxorubicin invivo was comparable in both models. Moreover, even in thesupposed doxorubicin-sensitive A2780 tumor model, a notice-able but not prolonged response was observed. These data sug-gested that a distinct higher intratumoral doxorubicin dose isrequired to achieve an effective and prolonged response in resis-tant tumors.

The results of the therapy study showed that application of a 7-fold doxorubicin equivalent dose of the pHPMA–doxorubicinconjugate triggered a very effective response in both resistanttumor types. In some mice, tumors completely disappeared.Furthermore, testing different treatment schedules indicated thatat least an overall dose of 5- or 6-fold doxorubicin equivalent wasnecessary to induce an effective response in these resistant tumors.Lower doses, that is, 3- to 4-fold, resulted in an incomplete tumorresponse, but still induced an improved inhibitory impact ontumor growth compared with free doxorubicin treatment. UsingFI, the pHPMA-conjugate mediated, improved intratumoraldoxorubicin accumulation could be proved. Application ofincreasing doses of pHPMA–doxorubicin conjugate resulted instepwise increased doxorubicin-derived signals. Even the 1-folddose led to a clear improvement compared with free doxorubicinwhich directly demonstrates the effect of the pHPMA-carrier.Together, from these observations, it was concluded that a suffi-cient high amount of doxorubicin can be delivered to and isreleased within the tumor tissue to kill resistant tumor cells.

The pattern of treatment-related response to the pHPMA–doxorubicin conjugate was observed to be different dependingon the tumor type. The onset of response of A2780cis tumorsgenerally occurred earlier compared with 1411HP tumors. In vitrocytotoxicity testing of pH-dependent drug release by pHPMA–doxorubicin conjugates suggested that xenograft specific charac-teristics are responsible for the different delayed responses ratherthan a pure cell-type dependent mechanism. Regarding the char-acteristic delayed response of 1411HP tumors, it was hypothe-sized that the microenvironment initially does not enable anefficient drug release within this tumor type. However, a time andtreatment-related switch to amore supportingmicroenvironmentseemed to occur resulting in a very effective release of doxorubicinfrom the highly accumulated pHPMA–doxorubicin conjugate.For proving the assumption, it was tested whether the microen-

vironmental switch can be displayed by the use of a hypoxia-indicating FI agent (Hypoxisense). The revealed data confirmedthe hypothesis and showed that the delayed response is closelyrelated with a switch to a more supporting microenvironment.Interestingly, a therapy-induced hypoxia when using HPMA-copolymerswas alreadydescribed.Minko and colleagues ascribedthis effect to the downregulation of VEGF gene expressioninduced by the HPMA copolymer doxorubicin conjugate (23).This could be an explanation for the phenomenon of the repro-ducible, time, and treatment-related effect observed in the1411HP model. In contrast, analyses with Hypoxisense agentrevealed that A2780cis tumors are characterized by a more sup-portingmicroenvironment which probably accelerates the releaseof doxorubicin from the conjugate resulting in an earlier response.

Drug carrier conjugates were applied with the aim to achievehigher intratumoral drug concentrations while protecting normaltissue thereby tackling the major obstacles in cancer chemother-apywhich are dose-limiting systemic toxicities and chemotherapyresistance. The new conjugate type investigated here, enables highintratumoral drug accumulation and overcomes resistance. How-ever, treatments at higher doses also induced some toxic sideeffects. As concluded from the in vitro experiments, a smallamount of doxorubicin is already released at pH 7.4 whichprobably leads to the dose-dependent toxicity in vivo due tocleavage of doxorubicin during circulation within the body. Inaddition, the toxicity profiles were different and seemed to bespecific to the particular tumor type. The higher toxicity observedin the A2780cis model might be associated with the more hyp-oxic/acidic microenvironment in these tumors. It can be specu-lated that a very early release of doxorubicin immediately afterdelivery of the conjugate in the tumor tissue may lead to arecirculation of a part of doxorubicin. This would also contributeto the occurrence of systemic toxicity.

The cleavage of the drug during circulationwithin the body anda possible premature release in the tumor tissuemight be reducedby a stabilization of the hydrazone bond. This could be accom-plished by applying a different spacer in the vicinity of thehydrazone bond. Chytil and colleagues developed and examinedpHPMA conjugates with different spacers and showed that thespacer had a distinct impact on the stability of the hydrazonebond (16). Thus, a refinement of the conjugate by stabilization ofthe hydrazone bond is assumed to further improve the character-istics of this system. Another point that needs to be optimized isthe clearance of these conjugates. Previous pharmacokinetic stud-ies were performed to determine the biodistribution, body circu-lation time, and elimination dependent onmolecular weight andarchitectural structure of the conjugates (15, 16, 24). The star-likepHPMA polymers with a molecular weight of 200 kDa whichleads to restricted renal elimination exhibited prolonged circula-tion and therefore much higher tumor accumulation comparedwith conjugates with lower molecular weight. Furthermore, aprevious study performed in our laboratory showed that after 3months, the amount of the 200 kDa pHPMA conjugate was belowdetection threshold indicating a hepatic/biliary elimination (15).Nevertheless, the elimination should be allowed within a shortertime due to renal excretion. Therefore, high molecular weight(star-like) HPMA copolymers that are degradable into lowermolecular weight units lying below renal threshold were alreadydeveloped. Thepresence of an enzymatically cleavable amino acidsequence (GFLG) acting as a spacer between the core and the sidechains of the star-like polymer conjugates enables the disassembly

Heinrich et al.





of the structure (25, 26). Thereby, an optimization of the currentpHPMA–doxorubicin conjugates will be performed in furtherstudies.

In conclusion, the current study demonstrates that the conceptof tumor site-restricted high-dose chemotherapy by usingpHPMA-drug conjugates is able to overcome therapy resistance.Further investigations in other tumor models and with relevantdrugs are important to prove the potential of this type ofconjugates.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: P. Chytil, T. Etrych, K. M€ader, T. MuellerDevelopment of methodology: A.-K. Heinrich, L. Schindler, K. M€ader,T. MuellerAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): A.-K. Heinrich, H. Lucas, L. Schindler, K. M€ader,T. MuellerAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): A.-K. Heinrich, H. Lucas, T. MuellerWriting, review, and/or revision of the manuscript: A.-K. Heinrich, L. Schind-ler, T. Etrych, K. M€ader, T. Mueller

Study supervision: T. Etrych, K. M€ader, T. MuellerOther (synthesis and physico-chemical characterization of polymer–drugconjugates): P. Chytil

AcknowledgmentsThe authors thankMrs. Franziska Reipsch for her excellent technical support.

Grant SupportThis work was supported by the project "BIOCEV – Biotechnology and

Biomedicine Centre of the Academy of Sciences and Charles University"(CZ.1.05/1.1.00/02.0109; to T. Etrych), from the European Regional Develop-ment Fund and by the Czech Science Foundation (project no. 15-02986S; toT. Etrych), by theGrant Agency of theCzech Republic Czech Science FoundationGACR (grant no. P207/12/J030 and 14-12742S; to T. Etrych), and also by theDeutsche Forschungsgemeinschaft DFG (MA 1648/8-1; to T. Mueller andK. M€ader).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received October 9, 2015; revised January 21, 2016; accepted January 21,2016; published OnlineFirst March 3, 2016.

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2016;15:998-1007. Published OnlineFirst March 3, 2016.Mol Cancer Ther Anne-Kathrin Heinrich, Henrike Lucas, Lucie Schindler, et al. Chemotherapy ResistanceTherapeutics with pH-Sensitive Drug Release Overcomes Improved Tumor-Specific Drug Accumulation by Polymer

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