Dual Fluorescent HPMA Copolymers for Passive Tumor Targeting with

Article pubs.acs.org/Biomac

Dual Fluorescent HPMA Copolymers for Passive Tumor Targeting with pH-Sensitive Drug Release: Synthesis and Characterization of Distribution and Tumor Accumulation in Mice by Noninvasive Multispectral Optical Imaging Stefan Hoffmann,† Lucie Vystrčilová,‡ Karel Ulbrich,‡ Tomás ̌ Etrych,‡ Henrike Caysa,§ Thomas Mueller,§ and Karsten Mad̈ er*,† †

Department of Pharmacy, Pharmaceutical Technology and Biopharmacy, Martin Luther University, Halle-Wittenberg, 06120 Halle, Germany ‡ Institute of Macromolecular Chemistry AS CR, v.v.i., Heyrovský Sq. 2, 162 06 Prague 6, Czech Republic § Department of Internal Medicine IV, Oncology/Hematology, Martin Luther University, Halle-Wittenberg, 06120 Halle, Germany S Supporting Information *

ABSTRACT: Preclinical in vivo characterization of new polymeric drug conjugate candidates is crucial for understanding the effects of certain chemical modifications on distribution and elimination of these carrier systems, which is the basis for rational drug design. In our study we synthesized dual fluorescent HPMA copolymers of different architectures and molecular weights, containing one fluorescent dye coupled via a stable hydrazide bond functioning as the carrier label and the other one modeling the drug bound to a carrier via a pH-sensitive hydrolytically cleavable hydrazone bond. Thus, it was possible to track the in vivo fate, namely distribution, elimination and tumor accumulation, of the polymer drug carrier and a cleavable model drug simultaneously and noninvasively in nude mice using multispectral optical imaging. We confirmed our in vivo results by more detailed ex vivo characterization (imaging and microscopy) of autopsied organs and tumors. There was no significant difference in relative biodistribution in the body between the 30 KDa linear and 200 KDa star-like polymer, but the star-like polymer circulated much longer. We observed a moderate accumulation of the polymeric carriers in the tumors. The accumulation of the pH-sensitive releasable model drug was even higher compared to the polymer accumulation. Additionally, we were able to follow the long-term in vivo fate and to prove a time-dependent tumor accumulation of HPMA copolymers over several days.

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enhanced permeability and retention (EPR) effect.11,12 The accumulation in solid tumors has been shown to be significantly increasing for increasing Mw of HPMA based drug delivery systems.13,14 Although efficient passive accumulation of polymer drug conjugates is a prerequisite for tumor-targeted therapy, the efficacy of treatment is also dependent on tumor specific drug release. In the past decade, we have shown that synthetic conjugates based on HPMA copolymers containing the cytotoxic drug doxorubicin (DOX) bound via pH-sensitive and hydrolytically degradable hydrazone bond are highly potent drug-delivery systems for chemotherapy.1,15 These DOXcontaining conjugates were stable in aqueous solution at a physiological pH of 7.4 (blood pH) but the drug was rapidly released in buffers at pH 5 - 6.5, simulating acidic pH in endosomes and lysosomes of cancer cells. Later on, highmolecular-weight (HMW) HPMA copolymer drug conjugates

INTRODUCTION In the last years, a considerable number of water-soluble synthetic polymers have been investigated as drug carrier systems suitable for delivery of conjugated therapeutics.1,2 Major advantages of drug conjugation with water-soluble polymers consist in reduced toxicity and side effects as well as in improved solubility, bioavailability, stability, circulation time, and biodistribution. 3 Polymer drug carriers based on N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers belong to the most intensively studied water-soluble synthetic polymers.4−7 Despite various applications have been investigated,8 most HPMA polymer drug conjugates were studied for the treatment of cancer, with a special focus on the sitespecific delivery of cytotoxic and anti-inflammatory agents into tumor tissues or cells.4,6 The potential of the polymeric prodrugs and especially of those intended for treatment of solid tumors could be significantly improved by increasing molecular weight (Mw) of the polymer carriers.9,10 Sufficiently high Mw of the polymer carrier may cause passive accumulation of the polymer drug conjugate in solid tumor tissues due to the © 2012 American Chemical Society

Received: October 25, 2011 Revised: December 23, 2011 Published: January 21, 2012 652

dx.doi.org/10.1021/bm2015027 | Biomacromolecules 2012, 13, 652−663

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anal. Calcd: C, 58.72%; H, 9.15%; N, 9.78%. Found: C, 58.98%; H, 9.18%; N, 9.82%. N-(tert-Butoxycarbonyl)-N′-(6-methacrylamidohexanoyl)hydrazine (Ma-ah-NHNH-Boc) was prepared in a two-step synthesis, as described previously29 (for more details, see Supporting Informations): Mp 110− 114 °C; purity (HPLC) > 99.5%. Elem. anal. Calcd: C, 57.70%; H, 8.33%; N, 13.46%. Found: C, 57.96%; H, 8.64%; N, 13.25%. 6-Methacrylamidohexanohydrazide (Ma-ah-NHNH2) was prepared in a two-step synthesis, as described previously30 (for more details, see Supporting Informations): Mp 79−81 °C. Elem. anal. Calcd: C, 56.32%; H, 8.98%; N, 19.70%. Found: C, 56.49%; H, 8.63%; N, 19.83%. DY-676-OPB derivative was prepared by modification of DY-676-NH2 by the thiazolidine-2-thione (TT)-activated 4-(2-oxopropyl)benzoic acid. DY-676-Amin (1.95 mg, 2.35 μmol) and OPB-TT (0.66 mg, 2,35 μmol) were dissolved each in 0.6 mL of a dimethylformamide−methanol (3:1) mixture. OPB-TT solution was added under stirring at 23 °C to DY-676NH2 solution simultaneously with 1 μL of 2.6 M NaOH solution (2.6 μmol). Course of reaction was monitored by TLC chromatography (eluent: metanol/ethylacetate/acetic acid 10:4:0.5): Rf DY‑676‑NH2 = 0.64, Rf DY‑676‑OPB = 0.87. The yield of the reaction was approximately 90% after 2 h. After 3 h, the reaction was stopped by evaporation of the solvent. HPLC showed one single peak at 11.38 min. Due to the very small amount of the product, the derivative was used immediately for the reaction with polymer precursors. Purity of all monomers and derivates was examined by 1H NMR (Bruker spectrometer, 300 MHz) and by HPLC (Shimadzu 10VP) using a C18 reverse-phase Chromolith Performance RP-18e (4.6 × 100 mm) column with diode array detection. The eluent was water− acetonitrile with a gradient of 5−95 vol % acetonitrile, 0.1% TFA, and a flow-rate of 1 mL/min. Synthesis of Polymer Precursors and Polymer−Dye Conjugates. Statistical copolymers of HPMA with Ma-ah-NHNH2 (precursor A) containing free hydrazide groups were prepared by radical copolymerization in methanol (AIBN, 0.8 wt %; monomer concentration 18 wt %; molar ratio HPMA/Ma-ah-NHNH2 93:7; 60 °C; 17 h) as previously described.30 The star polymer (precursor B) was synthesized by grafting the thiazolidine-2-thione (TT)-terminated semitelechelic HPMA copolymer (Mw = 26500 g/mol, Mw/Mn = 1.84) onto the second generation PAMAM dendrimers containing terminal amino groups.21 Semitelechelic copolymer precursor with chain-terminated TT groups (327 mg; 0.03 mmol TT groups) was dissolved in 8 mL of methanol and added under stirring into solution of 8 mg of D-NH2 (G2, diaminobutane core, 16 amino groups; 0.002 mmol of dendrimer) in 3.1 mL of methanol. After 2 h, the reaction was finished by adding 5 μL of 1-aminopropan-2-ol. Lowmolecular-weight impurities were removed by gel filtration (Sephadex LH20, solvent methanol). The polymer-modified dendrimer was isolated by precipitation in ethyl acetate. The free hydrazide groups needed for dye attachment to the polymer (precursor B) were obtained by removing the protective Boc groups from the hydrazides with concentrated TFA. Polymer conjugates A and B (Table 1, Scheme 1), bearing two fluorescent dyes, one attached via pH-sensitive hydrazone bond (model

with branched, grafted or star-like architecture and with pHsensitive drug release mechanism have been synthesized and tested for anticancer activity.16 Polymers with star-like architecture arouse particular interest in biomedical applications, and several structures have already been synthesized in the past decade.17−19 Higher molecular weights of such branched HPMA conjugates improved their therapeutic efficacy,10,20,21 which could be ascribed to higher accumulation of the polymers in tumor tissue due to the more pronounced EPR effect. However, the detailed in vivo fate of those drug delivery systems is not completely understood yet. Several studies were done to elucidate the in vivo fate of polymeric drug delivery systems, but the use of imaging techniques such as SPECT and PET allowed only an observation of the biodistribution for a few hours to days due to the short half-life of the used tracers.13,22,23 In this study, multispectral optical imaging has been used to follow the long-term in vivo fate of two HPMA copolymer-based drug delivery systems noninvasively in mice for several weeks. The use of two different fluorescent dyes in combination with multispectral optical imaging further allowed simultaneous characterization of the accumulation of the polymer carriers and of a pH-sensitive bond-coupled fluorescent model drug in solid tumors. Thus, it was possible to obtain simultaneous information on biodistribution from both the model drug and the polymer drug carriers. Optical imaging has been recently shown to be a versatile tool for tracking the in vivo fate of nanoscaled drug delivery systems.24−26 Major advantages of optical imaging compared to other imaging techniques are an easy setup (e.g., no radioactive labels), possible long-term observation even for several months, and also the possibility to observe two or more fluorescent probes simultaneously. We synthesized two different HPMA-based polymer drug carrier systems, linear and star-like HMW polymers, each containing two fluorescent probes (DY-676, DY-782). The use of NIR and Far Red fluorescent dyes enabled us to obtain information about the distribution also from deep tissues like liver, spleen, and kidneys.27 One dye was coupled via a pHsensitive hydrolytically degradable hydrazone bond (model drug), whereas the other one was coupled with the copolymer carrier backbone via a noncleavable hydrazide bond. Aims of the current study were to observe the body fate of the polymer and the model drug noninvasively and simultaneously for several weeks in healthy and tumor-bearing mice using multispectral optical imaging and to investigate the impact of the polymer structure on the biodistribution and accumulation in a xenograft tumor model. A long observation time would clearly be an advantage over other studies that were limited to a few hours.13,22,23

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MATERIALS AND METHODS

Table 1. Physicochemical Characteristics of Polymer Precursors and Dye-Coupled Polymers

Chemicals. 1-Aminopropan-2-ol, methacryloyl chloride, 2,2′-azobis(isobutyronitrile) (AIBN), 6-aminohexanoic acid (ah), N,N′-dimethylformamide (DMF), N,N′-dicyclohexylcarbodiimide (DCC), N-ethyldiisopropylamine (EDPA), dimethyl sulfoxide (DMSO), tert-butyl carbazate, ethylenediaminetetraacetic acid (EDTA), 4-(2-oxopropyl)benzoic acid (OPB), trifluoroacetic acid (TFA), dimethylaminopyridine (DMAP), and 2,4,6-trinitrobenzene-1-sulfonic acid (TNBSA) were purchased from Fluka. Doxorubicin hydrochloride (DOX-HCl) was purchased from Meiji Seiko, Japan. Poly(amido amine) (PAMAM) dendrimers were purchased from Dendritic Nanotechnologies, Inc., U.S.A. Fluorescence dyes DY-782-NHS-ester and DY-676-NH2 (aminomodified) were purchased from Dyomics GmbH, Germany. Synthesis of Monomers and Derivatives of DY-676. N-(2Hydroxypropyl)methacrylamide (HPMA) was synthesized as described previously28 using K2CO3 as a base (for more details, see Supporting Informations): Mp 70 °C; purity > 99.8% (HPLC). Elem.

polymer precursor A precursor B A B

Mw (g/mol)

Mw/ Mn

Rh (nm)

27200 186000

1.74 1.76

4.3 12.7

DY-782 (wt %)

DY-676 (wt %)

1.6 1.4

0.7 0.7

drug) and the second through a stable hydrazide bond (polymer chain label), were prepared by the consecutive reactions of the polymeric precursors with DY-782-NHS-ester and derivative DY-676-OPB. Polymer precursor A (100 mg) and DY-782-NHS-ester (2 mg, 2.02 μmol) were dissolved each in 0.5 mL of dimethyl acetamide (DMA). Into the stirring solution of polymer precursor were added at one time DMAP (1 mg, 8.1 μmol) and the DY-782-NHS-ester solution. The course of the reaction 653


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Helios α spectrophotometer (Thermochrom). Molar absorption coefficients of the DY-782 (ε782 = 170000 L mol−1 cm−1 (ethanol)) and DY-676 (ε676 = 180000 L mol−1 cm−1 (ethanol)) were used for calculation of the dye content. Mass spectrometry (MS) was measured on LCQ Fleet instrument (Thermo Scientific) using ESI ionization. The size distribution (hydrodynamic radius (Rh)) in a phosphate buffer (0.1 M, with 0.05 M NaCl; polymer concentrations: 0.01 g/mL) at 25 °C were measured by dynamic light scattering (DLS) with a Nano-ZS instrument Zetasizer (ZEN3600, Malvern). For evaluation of DLS data, the DTS(Nano) program was used. The values were a mean of at least five independent measurements. In Vitro Release of Model Drug (DY-676). The rate of DY-676 release from polymers A and B (polymer concentration equivalent to 16 μM DY-676) was investigated in phosphate buffers at pH 5.0 or 7.4 (0.1 M phosphate buffer with 0.05 M NaCl) at 37 °C using GPC in aqueous methanol solution (Shimadzu HPLC system equipped with UV−vis detector (Shimadzu SPD-10AVvp); eluent was methanol− sodium acetate buffer (80:20 vol %) for the TSKgel G3000SWxl column; 0.5 mL/min). The content of released DY-676 was calculated from the area of the corresponding peaks (free DY-676 peak/free DY676 peak + polymer-DY-676 peak) at λ = 676 nm. All release data are expressed as the amounts of free DY-676 to its total content in the conjugate. All experiments were carried out in triplicates. Animal Care and In Vivo Multispectral Optical Imaging. All in vivo experiments complied with regional regulations and guidelines and were approved by the local authority in Saxony-Anhalt. The mice were kept under controlled conditions (12 h day/night cycle, 24 °C).

was monitored by TLC chromatography (eluent: methanol/ethyl acetate/ acetic acid 10:4:0.5): Rf DY‑782‑NHS‑ester = 0.84, Rf polymer = 0. After 2 h at room temperature, TLC showed approximately 85% yield of the reaction and polymer bearing DY-782 was purified from low-molecular impurities by gel filtration (Sephadex LH-20, solvent methanol) and isolated by precipitation in ethyl acetate. Prior the attachment of DY-676-OPB dye, the polymer containing DY-782 (80 mg) was dissolved in 0.8 mL of methanol and added to the solution of DY-676-OPB (1.03 mg, 1.04 μmol) in 0.2 mL of methanol together with acetic acid (40 μL/mL of methanol). Course of reaction was monitored by TLC chromatography (eluent: methanol/ethyl acetate/acetic acid 10:4:0,5): Rf DY‑676‑OPB = 0.75, Rf polymer = 0. After 12 h, approximately 70% of DY-676-OPB was attached to the polymer that was purified by gel filtration (Sephadex LH-20, solvent methanol) and isolated by precipitation in ethyl acetate. The content of hydrazide groups was determined by a modified TNBSA assay as described earlier.31 Molar absorption coefficient ε500 = 17200 L mol−1 cm−1 estimated for the model reaction of Ma-ah-NHNH2 with TNBSA was used. Determination of molecular weight and polydispersity of all prepared polymers and the content of free polymer in star polymer precursors or conjugates were determined by GPC/ HPLC Shimadzu system equipped with UV−vis (Shimadzu SPD10AVvp), refractive index Optilab-rEX, and multiangle light scattering DAWN EOS detectors (both Wyatt Technology Co.). The eluent was 0.3 M sodium acetate buffer pH 7.4 for the Superose 6 HR 10/30 column and methanol−sodium acetate buffer (80:20 vol %) for the TSKgel G4000SWxl column; flow rate 0.5 mL/min. The total content of the dyes in polymer conjugates was determined spectrophotometrically on a

Scheme 1. Structures of Linear (A) and Star-Like (B) HPMA Copolymer−Dye Conjugates

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Figure 1. (a) Release of model drug (DY-676) from linear and star like HPMA copolymer incubated in phosphate buffered saline at pH 5 and pH 7 at 37 °C, data represent mean ± SD. (b) Emission spectra of DY-676, DY-782 and autofluorescence spectrum of SKH-1 mice. Elimination of (c) cleavable model drug and (d) HPMA copolymers after intravenous administration of 1 mg polymer to healthy SKH-1 mice (n = 3, data represents mean ± minimum and maximum value). For the imaging process, mice were anesthetised using an initial dose of 2.5% isoflurane for veterinary use in oxygen at a flow of 2 L per minute and a continuous dose of 2% isoflurane in oxygen (2 L/min). During the anesthesia mice were placed on a tempered plate (35 °C) to avoid decrease of body temperature. Fluorescence imaging was carried out in the Maestro in vivo imaging system from CRi (Cambridge Research and Instrumentation, U.S.A.). Two filter sets were combined to acquire one image cube in the range of 680 to 950 nm containing both emission signals from DY-676 and DY782 (“Red”: excitation filter, 615−665 nm; emission filter, 700 nm longpass; and “Near Infrared”: excitation filter, 710−760 nm; emission filter, 800 nm long-pass). All images were automatically exposed to ensure highest information content. The Maestro software (version: 2.10.0) was used to separate the spectral species from the cube (Figure 1b) and to calculate unmixed greyscale images of single components (DY-676 and DY-782), which can be overlaid afterward to a mixed component image. The greyscale images were intensity weighted and displayed using the hot color profile. Unfortunately, it was not possible to subtract the mouse autofluorescence spectrum from the images because the spectrum was too similar to the combined spectrum of both dyes (Figure 1b). Mouse autofluorescence is negligible in near-infrared area of the spectrum (>800 nm) but in the red area there is still a mouse autofluorescence signal, contributing approximately 5% to the DY-676 signal. Distribution and elimination of the new HPMA copolymers was first investigated in healthy nude female mice (SKH1-Hrhr) from Charles River Lab (2−4 month old). A total of 10 mg of each polymer was dissolved in 1 mL of isotonic sorbitol solution and sterile filtered (0.2 μm Millex, Millipore, U.S.A.). Eight mice were randomized into two groups, and each mouse was intravenously injected into the tail with 100 μL of the polymer solution (according 1 mg polymer). A total of 24 h after administration, one mouse from each group was sacrificed and autopsied to gather more detailed information about the distribution of the polymers in mouse organs. Investigation of tumor accumulation was performed in

human xenograft colon carcinoma models (DLD-1 wild-type and HT-29). Athymic mice were ordered from Harlan Winkelmann, Germany (Hs1Cpb:NMRI-Foxn1nu, 5 weeks old, male). After a 2 weeks setting in period, mice were short-term anesthetized using isofluorane, and tumor cells suspended in 150 μL of PBS were subcutaneously injected to the left (HT-29, 5 × 106 cells) and right (DLD-1, 5 × 106 cells) side of the mice. Mouse weight and tumor size was measured every 2−3 days. The tumor volume (V) was estimated based on length (l) and width (w) using the equation

π V = l × w2 × 6 according to the results of Euhus et al. and Tomayko et al.32,33 Mice were randomized into 2 groups at day 19 after tumor injection according to their tumor size. The tumor sizes in both groups were comparable (Figure 3a,b). A total of 21 days after tumor cell injection, the palpable tumors in both groups had an average volume of 0.85 ± 0.22 cm3 (DLD-1) and 0.55 ± 0.21 cm3 (HT-29), and the polymer solutions were intravenously injected into the tail (100 μL of isotonic sorbitol solution containing 1.5% polymer, sterile filtered before injection). After 6 days, the same dose was administered again. Two days after the second injection, all mice had to be sacrificed due to tumor burden. Ex Vivo Fluorescence Imaging of Autopsied Organs. To gather more detailed information about the distribution of the polymers and model drug in mouse organs, one healthy mouse was sacrificed 24 h after intravenous administration of 1 mg of polymer. Further, the organs of all tumor-bearing mice were investigated after they had to be sacrificed. The organs were autopsied and placed in a 24-well plate for fluorescence imaging. The measured intensities were normalized by the organ area and exposure time to be comparable within the two groups. As a strong accumulation in specific areas of the kidney could be observed, the kidneys were investigated in more detail by confocal laser scanning microscopy (LSM 710 from Zeiss; excitation, 633 nm HeNe Laser). 655


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Figure 2. Distribution of HPMA copolymers and cleavable model drug in SKH-1 mice 6 h after i.v. administration of 1 mg linear HPMA (30 KDa, polymer A) or star-like HPMA (200 KDa, polymer B) in dorsal and abdominal images. Arrows mark bladder (black), kidneys (white) and intestine (blue).

Figure 3. Tumor volume in both groups is comparable: (a) linear HPMA 30 kDa, (b) star-like HPMA, 200 kDa; data represents mean ± minimum and maximum value. A reliable tumor accumulation value that is based on total fluorescence intensity of the tumor area (d) and on the total intensity of the whole mouse (e) was calculated from the unmixed image (c). Histological Characterization of Autopsied Tumors. The autopsied tumors were sliced and regions of strong and low model drug fluorescence were fixed in 4% paraformaldehyde, embedded in paraffin, sliced (4 nm), dewaxed, and stained with hematoxylin and eosin to be observed by light microscopy.

nondegradable hydrazide bond or pH-sensitive hydrazone bond (cf. Scheme 1). With the aim to introduce a reactive keto group into the structure of DY-676-NH2 we acylated the dye amino group with thiazolidine-2-thione amide of OPB acid. Purity of product was 92% (determined by HPLC analysis with UV detection) and the structure was confirmed by MS (ESI): m/z 988.71 [M + H]+. The precursor of polymer A (cf. Table 1) was prepared by radical solution copolymerization of HPMA with MA-ahNHNH2 using AIBN as initiator and methanol as solvent. The average Mw of polymer precursor A was determined to be 27200 g/mol and it contained 5.7 mol % of hydrazide groups, a content sufficient for attachment of both fluorescent dyes to the polymer. We assume that this polymer is still excretable from

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RESULTS AND DISCUSSIONS Synthesis of Polymer−Dye Conjugates. A linear HPMA copolymer of 30KDa (polymer A, Scheme 1) and a star-like HMW polymer of 200 kDa (polymer B, Scheme 1) were investigated with regard to their biodistribution, elimination, and tumor accumulation. Two fluorescent dyes with different emission wavelength maximum in the near-infrared (NIR) and far red (FR) area of spectrum were coupled to HPMA copolymer precursors A or B via 656


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the star-like polymer (B). After 46 h, the signal decreased to ∼5%, which is the autofluorescence level of the mice in this area of the spectrum. It can be concluded that the model drug was completely eliminated 2 days after injection and its elimination is controlled by the rate of its release from the polymer carrier during circulation. The elimination of the model drug could be fitted for both polymers assuming a first order exponential decay. The calculated half-life was comparable for both polymers (0.3 days for both polymers, Figure 1c). The polymer structure itself had absolutely no influence on the body distribution, but the elimination of the polymers was dependent on polymer architecture and size, as it could be expected from other studies.34,36 Whereas the fluorescence intensity of the noncleavable dye (DY-782) from polymer A decreased below 50% of the initial intensity after 1 day (48.5%), 14 days were required for polymer B until the remaining fluorescence intensity decreased below this value (48.4%, Figure 1d). As expected, the circulation time was much longer for the HMW star-like polymer. This can be explained by the much higher average molecular weight of the star like polymer (∼200 kDa) and more rigid polymer skeleton. Whereas the smaller linear polymer is still below renal elimination threshold, the star-like polymer cannot be excreted by the kidneys. The elimination kinetic of both polymers could be fitted assuming a second order exponential decay that can be explained with both possible elimination pathways for the HPMA copolymers: renal (mainly in the first hours after injection and for the small molecular weight fraction) and hepatic clearance of the polymer. As the synthesized HPMA copolymers are not biodegradable, we assume a major influence of hepatic elimination, especially for the star-like polymer B. After injection, both polymers immediately distributed all over the mouse body. Already 5 min after injection, an intensive fluorescence signal from both dyes (polymer attached and cleavable model drug) was detectable in the bladder, indicating renal elimination. In the case of the star-like HMW polymer the bladder fluorescence could be ascribed to the small portion (up to 15%) of semitelechelic polymer, which was not grafted to the central dendrimer core. After 2 h, an intensive signal of the model drug was also detectable from the gut. As there was no signal for the polymer-attached fluorescent dye (DY-782) from the gut, it can be concluded that the model drug is quite rapidly splitted off the polymer and that it is excreted via hepatic and renal elimination pathways. Interestingly, there was no particular accumulation in liver and spleen detectable compared to other organs, which is a major advantage over most nanoparticular drug delivery systems that often show very strong accumulation in liver and spleen due to an uptake of the drug delivery systems by the reticuloendothelial system.24,37,38 Both polymers showed intensive accumulation in the kidneys even already 5 min after injection. To exclude the possibility of a specific accumulation of the fluorescent dyes, three mice were treated with free DY-676-OPB and free DY-782 (control). Both dyes were rapidly excreted and the fluorescence intensity decreased to the autofluorescence level of the mice within 26 h (data not shown). It can be concluded that the kidney accumulation is a property of the investigated HPMA copolymers. No mouse showed any pathologic effects from this accumulation. Interestingly, in dorsal images a polymer accumulation in kidneys was still observable, when the abdominal images did not show any accumulation in the bladder. This indicates a specific interaction between polymer and kidneys. The signal of polymer remained in the kidney despite the fact that no polymer was filtrated through the kidney to bladder.

organism by glomerular filtration. The star polymer precursor B was synthesized by grafting TT group-terminated semitelechelic HPMA-based polymer onto the PAMAM dendrimer containing amino groups. The products of the grafting reaction was a water-soluble HMW polymer of a star-like structure, which made it suitable as drug carrier for passive targeting to solid tumors.16 The Rh of the polymer coil of the star polymer precursor B in aqueous solution was 12.7 nm, which is 3-fold higher than that of linear polymer precursor A (4.3 nm). The increase in Mw fulfilled the prerequisite criteria for enhanced accumulation of polymers in solid tumors due to the EPR effect.34 Removing the protective Boc groups from the hydrazides in polymer precursors with TFA did not change molecular characteristics. Attachment of both fluorescent dyes was carried out consecutively in DMA and thereafter in methanol solution of polymer precursors. First, DY-782 was conjugated with the copolymer within 2 h in high yield (85%) via stable hydrazide bond by the hydrazinolysis of NHS-ester of DY-782-NHS by hydrazide groups of appropriate polymer carrier. Consequently, the second dye (DY-676-OPB) was attached to the copolymer by a hydrazide-ketone condensation reaction in methanol in the presence of acetic acid (40 μL/mL). Attachment of the DY-676 derivative to the polymer precursor was considerably slower; within 12 h around 70% of the dye derivative was attached to the polymer precursor. For both polymer architectures, attachment of the fluorescent dyes had no distinct influence on the distribution of molecular weights of polymer carriers. However, we were unable to determine the exact Mw and Rh of the polymer conjugates after conjugation of DY-676 and DY-782 due to the interference of dye fluorescence with the lasers used in dynamic light scattering. In Vitro Model Drug Release. There are several articles describing polymeric drug delivery systems with pH-controlled release of active molecules.1,16,35 Prerequisite for these systems is a different release rate of the active molecule in neutral and slightly acidic environment; relative stability at pH 7.4 and a fast release at pH 5−6.5. To verify the stability of polymer conjugates in vitro, studies of DY-676-OPB release from HPMA copolymers were carried out in phosphate buffered saline at pH 7.4 and 5.0, simulating conditions in blood and in endosomes of target cells respectively (Figure 1a). As expected, results of pH-dependent chemical hydrolysis at 37 °C showed a significant difference in the model drug release (DY-676), with the rate at pH 5 being much higher than that observed at pH 7.4 (76% of DY-676-OPB released within 1 h at pH 5 in comparison with only 12% released in a buffer of pH 7.4). There was no significant difference in the release rates from the carriers differing in polymer architecture. Unfortunately, the rate of model drug release at pH 7.4 was noticeably higher than the rate of doxorubicin release from similar polymer drug carriers.31 There was no hint for any release of the polymer chain label DY-782 during the incubation of the conjugate in the buffers, proving the stability of the hydrazide bond between DY-782 and the polymer carrier. In Vivo Fluorescence Imaging in Healthy Mice. The distribution and elimination of both polymers was investigated in nude female mice (SKH1-h hr) over 80 days. The fluorescence image cubes were processed as described in the Materials and Methods. Typical intensity weighted fluorescence component images are presented in Figure 2. The fluorescence intensity of the cleavable model drug decreased independently from the polymer architecture. A total of 22 h after injection, the fluorescence intensity decreased to 9.5% of the highest measured intensity for the linear polymer (A) and 12.3% for 657


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based on reliable numbers. However, using this method in combination with NIR-fluorescent dyes the accumulation in tumors is even underestimated, because NIR light is diffuse scattered inside the tissues rather than being absorbed.27 The more intensive fluorescence signal from the tumor is also scattered to the whole mouse body, so that the mouse body seems to be brighter than it is. Therefore we expect even better accumulation of the HPMA copolymers than we calculated. The distribution and elimination of the HPMA carrier polymers and of the model drug (DY-676) were basically comparable with the data observed in healthy mice. Both polymers accumulated in the tumors. The calculated relative tumor accumulation (TAV) of the HPMA copolymers was comparable and was not dependent on the polymer architecture (Figure 4) but the accumulation of the cleavable dye was always higher for the star-like polymer (B). Over the time the tumor accumulation of polymers increased more and more (Figure 4a−d). For example, 4 h after injection, the TAV in the group of the linear 30 kDa polymer (A) was calculated to be 1.6 ± 0.10 in DLD-1 and it increased to 2.48 ± 0.7 during the next 5 days. This can be explained with the EPR effect: as the polymer is long circulating in the blood, a small fraction of it can always diffuse into the tumor and is retained there. The pH-sensitive coupled model drug (DY676) showed a much stronger accumulation in the tumors, particularly in DLD-1, than the polymers (2 days after injection of the linear polymer (A) 4.51 ± 0.40 for DLD-1 and 2.66 ± 1.30 for HT-29; 2 days after injection of the starlike polymer (B) 7.20 ± 1.8 for DLD-1 and 3.48 ± 0.54 for HT-29), but opposite to the polymers there was an optimum accumulation 2 days after injection, and afterward, the TAV decreased due to rapid elimination of the small molecule (Figure 4e). We suppose that due to an acidic microenvironment in the tumors the model drug is cleaved in the tumors and can diffuse inside the tissue. However, after 2 days, most of the cleavable dye was already excreted and the overall amount in the tumor was rather small. Administration of a second dose of polymer after 6 days even led to increased tumor accumulation of the model drug because there was still model drug retained in the tumor. However, even better accumulation would be achieved if the model drug is released slower in the bloodstream, because the accumulation of the polymer took much more time than the release of the model drug. Generally, a better accumulation of the model drug could be observed in DLD-1 compared to HT-29. Ex Vivo Organ and Tumor Characterization. Organs of tumor-bearing mice were excised immediately after sacrificing the mice 2 days after administration of the second polymer dose due to tumor burden. At this time, the tumors had an average volume of 1.35 ± 0.23 cm3 (DLD-1) and 0.93 ± 0.20 cm3 (HT-29) and the mice had already lost about 10% of their weight. Tumors and other important organs were placed in a 24-well plate and imaged using the Maestro imaging system and automatic exposure function. An exemplary image is presented in Figure 5a,b. The model drug signal is almost exclusively detectable from both tumors, whereas the polymer signal is also measured in all other organs. The distribution of HPMA copolymers in organs of tumorbearing athymic nude mice was comparable with the distribution in organs of healthy mice (SKH-1). The fluorescence signal from the organs was normalized by exposure time and organ area to

The accumulation of the polymers is located mainly in the cortex of kidney (Figure 5d−f) and has been already reported for HPMAbased polymers.13,22,23 Most probably, it could be ascribed to the interaction of polymer with the glomerular basement membrane in the cortex. Hydrazide groups of the polymer can interact with the highly anionic membrane, which is composed of a collagen based network containing laminin, entactin, and proteoglycan (with high heparin and chondroitin sulfate content).39 In Vivo Fluorescence Imaging in Tumor-Bearing Mice: Tumor Accumulation. Passive tumor accumulation using HMW polymeric drug carriers due to the EPR effect of tumor vasculature has been first described more than 25 years ago11 and is a well-accepted approach today.12 Due to the long circulation time in healthy mice we expected a considerable accumulation of the new HPMA copolymers in solid tumors in mice, based on the enhanced permeability of some of the blood vessels. To investigate this effect, human xenograft colon carcinomas (DLD-1 and HT-29) have been successfully inoculated in athymic mice. These tumors are characterized by a lot of necrotic areas due to insufficient vascularisation in our model and therefore are suspected to have acidic microenvironment due to enhanced metabolism. Thereby we also expected enhanced cleavage of the acid-sensitive hydrazone-bond locally inside the tumors. Further, the extravasated HPMA copolymer−drug conjugate can be internalized by the tumor cells,40 and the model drug should be cleaved in endosomes and lysosomes. Both processes should result in a local accumulation of the model drug in the tumors. Xenograft tumors were inoculated and after 21 days both polymers were intravenously injected at a concentration of 1.5 mg per mouse (n = 3 per group). At this time the solid and palpable tumors had already a volume of 0.85 ± 0.22 cm3 (DLD-1) and 0.55 ± 0.21 cm3 (HT-29). The mouse weight was continuously checked and decreased due to tumor burden. Weight and tumor size were comparable in both groups (Figure 3a,b). A second dose of the polymers (1.5 mg per mouse) was administered after 6 days. A total of 48 h after injection of the second dose, all mice had to be sacrificed due to the tumor burden. At this time the mice lost about 10% of their weight. To analyze and compare the tumor accumulation of the HPMA copolymers and model drug, a special calculation method was developed (Figure 3c−e). Applying this method, a comparable “Tumor Accumulation Value” (TAV) could be calculated from the unmixed greyscale images of the single spectral species, based on fluorescence intensity (I) and fluorescent area: TAV =

proportion of fluorescence signal proportion of fluorescence area

Itumor Imouse − Itumor area tumor proportion of fluorescence area = area mouse − area tumor proportion of fluorescence signal =

× (area mouse − area tumor) I TAV = tumor area tumor × (Imouse − Itumor)

The advantage of this calculation method is that the average fluorescence signal from the region of the tumor is compared to the average fluorescence signal of the mouse body without tumor. Thus, intensity fluctuations and differences within different regions of the tumor could be eliminated. Using this calculation method, the accumulation of different polymers or in different tumors can be compared 658


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Figure 4. (a−d) Time-dependent change of tumor accumulation of model drug and polymer drug carriers in DLD-1 and HT-29 xenograft model after administration of 1.5 mg polymer at the beginning and after 145 h. (e, f) Tumor accumulation values 2 days after first (e) and 2 days after second (f) dose. All data represent mean ± minimum and maximum.

detectable in all investigated organs. A very high accumulation was found in the kidneys but also in both tumors. Kidney accumulation of HPMA copolymers was also found by other groups before,13,22,23 but no toxic effects are known to arise from this accumulation. Interestingly, the accumulation of the polymeric drug delivery systems in the liver and spleen was rather small compared to kidneys and tumor. This is an advantage over many other nanoscaled drug delivery systems that often show tremendous and undesired accumulation in liver and spleen due to uptake by the reticuloendothelial system.24,37,38 The relative distribution of both HPMA copolymers in the body is comparable, but the star-like architecture of polymer B results in higher accumulation of the polymer in the tumors, which is based on the enhanced EPR effect for HMW polymers. However, the increased extravasation to the tumor did not result in a higher model drug concentration, which can be explained by rapid cleavage of the dye. While significant accumulation of polymer in tumors could be found after 12/24 h, in the same time interval, more than 50−75% of model drug is already released. It is likely that

be comparable. The fluorescence intensities from different organs are displayed in Figure 6a−d and are basically in good agreement with the in vivo data. The relative distribution of DY-676 is the same for both HPMA copolymers and, thus, independent of the polymer architecture. As it was already known from the in vivo images, the highest model drug signal is detectable from the tumors, although the variability is rather high. The model drug is retained in the tumor tissue, whereas it is much more rapidly cleared from the blood and from other organs (Figure 6a,b). The smaller fluorescence in lung, testes, gallbladder, and gut compared to kidneys, liver, spleen, and heart can be explained by the lower blood content in these organs, leading to a decreased light absorption compared to the well blood supplied organs. However, a very intensive fluorescence signal is detectable from the kidneys when they are autopsied already 24 h after administration of the polymer (Figure 5c) due to renal excretion of the released model drug and interaction of polymer with glomerular basement membrane in kidney cortex, as already discussed above. The HPMA copolymer signal is 659


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Figure 5. (a, b) Organ images of model drug DY-676 (a) and HPMA copolymer (b) of a mouse that was treated with star-like HPMA copolymer. (c) Distribution of model drug in kidneys 24 h after intravenous administration: left, placebo; middle, star-like HPMA copolymer; right, linear HPMA copolymer. (d−f) Pseudo-colored fluorescence images of kidney slices 24 h after i.v. injection: model drug, blue; HPMA copolymer, yellow (linear HPMA copolymer, d and e; star-like HPMA copolymer, f). (g) Confocal microscopic images of model drug distribution in the kidney 24 h after i.v. injection of 1.5 mg linear HPMA copolymer (polymer A).

Figure 6. (a−d) Distribution of model drug and HPMA copolymers in mouse organs 49 h after injection of 1.5 mg polymer [a, c: linear HPMA copolymer 30 kDa (A); b, d: star-like HPMA copolymer, 200 kDa (B)]. All data are mean ± minimum and maximum values.

the dye was cleaved before the polymeric carrier could accumulate in the tumor, and thus, using a more stable pH-sensitive coupling of the model drug, decreasing the release rate, would probably also positively influence the distribution of the model drug. As it could be seen in images of autopsied kidneys 24 h after injection of 1 mg polymer to female mice (SKH-1), a high fluorescence

signal of the model drug is particularly detectable from the pyramids, whereas the polymers themselves were more homogeneously distributed in the outer area of the kidneys (Figure 5d−f). The concentration of the model drug in the loop of Henle due to excretion was proven by confocal laser scanning microscopy (Figure 5g, placebo kidney under the same conditions 660


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Figure 7. Fluorescence images of autopsied tumors (cross section) 49 h after administration of the second dose of polymers. An inhomogeneous distribution of model drug and homogeneous distribution of polymers inside the tumor was observable.

Figure 8. (a−d) Microscopic sections of the tumors from a mouse that was treated with polymer A (30 KDa, linear) from a region of low model drug intensity (a, c) and from a region of high model drug intensity (b, d). Sections from regions of high model drug fluorescence are characterized by only few living cells (marked by arrows) and large necrotic areas, whereas sections from regions of low model drug fluorescence show mainly vital cells.

from regions of the tumors that showed high or low model drug fluorescence. Whereas the slices from tumor regions of low model drug fluorescence showed almost exclusively tissues of living tumor cells (Figure 8a,c), large necrotic areas with few living tumor cells were visible in the slices of intensive fluorescent tumor regions (Figure 8b,d). An enhanced accumulation of the model drug in necrotic areas of the tumors is evident and we suppose that the local microenvironment (pH) in these areas increases the rate of model drug release.

showed no fluorescence signal). For a detailed characterization, the autopsied tumors were sliced and imaged with higher magnification (Figure 7). It was obvious that the model drug was nonhomogeneously distributed and concentrated in central regions of the tumors (both cell lines), whereas the HPMA copolymers were homogeneously distributed in the whole tumor. Also, the results showed a better accumulation of the model drug in DLD-1 tumors compared to HT-29. For more detailed microscopic histological characterization, small pieces were cut 661


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CONCLUSION In our study, we characterized the distribution and elimination of those polymeric drug carrier systems conjugated with a fluorescent and cleavable model drug noninvasively in vivo. In contrast to other studies, we were able to track the in vivo fate for several weeks and to observe the distribution of the polymer and a conjugated model drug simultaneously by multispectral optical imaging. For that purpose, HPMA copolymers differing in polymer architecture, linear and star-like, were successfully coupled with two fluorescent probes with different emission properties. We could not detect any difference in the relative biodistribution in the body between the 30 KDa linear and 200 KDa star-like polymer, but the star-like polymer circulated much longer. Although the synthesized polymers were not biodegradable, even the 200 KDa star-like polymer was completely eliminated within 3 months. Further we investigated the tumor accumulation in solid human colon carcinoma xenograft models in vivo in athymic nude mice. A new calculation approach was developed to compare the accumulation of different polymers in tumors based on a reliable method. The polymeric carriers accumulated in the tumors as well as the cleavable model drug. The polymer accumulation was quite slow. It took more than 24 h to reach a plateau. This time-dependent accumulation of the polymeric carrier emphasizes the importance of noninvasive long-term biodistribution and tumor accumulation studies. Moreover, the accumulation of model drug (cleavable dye) was smaller than it was already reported for doxorubicin in comparable HPMA copolymers.10,16,21 Most probably, this might be impact of the overly fast rate of model drug release or, less possibly, a tumor cell line-dependent phenomenon. Low accumulation of HPMA copolymers was already reported for AT1 prostate cancer,22 whereas very high accumulation of HPMA copolymer delivered doxorubicin was found in EL4 T-cell lymphoma.16 We can conclude that not only the distribution of the polymer carrier plays a key role in the delivery of the active drug to the tumors, but also the proper selection of the biodegradable spacer between polymer carrier and active drug. We suppose that a much better accumulation of the model drug in the tumors is presumable when the rate of release in blood environment is decreased and we will investigate this in further studies. The in vivo data could be confirmed by ex vivo imaging of the extracted organs. Further, we were able to show that the accumulation of the pH-sensitive cleavable model drug is situated in local, necrotic areas of the tumors and we suppose that this effect is caused by the local acidic microenvironment in the tumors and will confirm the local acidic pH in follow-up studies.

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by the Grant Agency of the Czech Republic (Grant No. P301/ 11/0325), and by the Deutsche Forschungsgemeinschaft DFG (MA 1648/7-1 and LSM: INST 271/250-1). Martina Hennicke and Constanze Gottschalk are acknowledged for the animal care.

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ASSOCIATED CONTENT

S Supporting Information *

Detailed description of the synthesis of the monomers for polymerization. This material is available free of charge via the Internet at http://pubs.acs.org.

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REFERENCES

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected].

ACKNOWLEDGMENTS This work was supported by the Grant Agency of the Academy of Sciences of the Czech Republic (Grant No. IAAX00500803), 662


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Dual Fluorescent HPMA Copolymers for Passive Tumor Targeting with

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