COREL-07352; No of Pages 8 Journal of Controlled Release xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

Journal of Controlled Release journal homepage: www.elsevier.com/locate/jconrel

a

8

a r t i c l e

9 10 11 12

Article history: Received 3 July 2014 Accepted 19 August 2014 Available online xxxx

13 14 15 25 16 17

Keywords: Drug delivery FRET imaging Intracellular release Polymer therapeutic

c

Institut für Chemie und Biochemie, Freie Universität Berlin, Takustrasse 3, Berlin 14195, Germany Leibniz-Institut für Molekulare Pharmakologie (FMP) & Freie Universität Berlin, Robert-Roessle-Str. 10, Berlin 13125, Germany mivenion GmbH, Robert-Koch-Platz 4, Berlin 10115, Germany

i n f o

a b s t r a c t

Herein we present a FRET-based theranostic macromolecular prodrug (TMP) composed of (a) dendritic polyglycerol (PG) as polymeric nanocarrier, (b) Dox linked via a pH-sensitive hydrazone to (c) a tri-functional linker, and (d) an indodicarbocyanine dye (IDCC) attached in close proximity to Doxorubicin (Dox). The drug fluorescence is quenched via intramolecular FRET until the pH-sensitive hydrazone bond between the TMP and Dox is cleaved at acidic pH. By measuring its fluorescence, we characterized the TMP cleavage kinetics at different pH values in vitro. The intracellular release of Dox from the carrier was monitored in real time in intact cancer cells, giving more insight into the mode of action of a polymer drug conjugate. © 2014 Published by Elsevier B.V.

29 27 26

E

1. Introduction

31

Theranostic nanomedicine has emerged as a field of research with great potential for the simultaneous detection and treatment of various diseases such as cancer and cardiovascular dysfunctions. [1] The functional combination of drugs and imaging agents within theranostic nanocarriers enables diagnosis, drug delivery, and monitoring of therapeutic response. It has been suggested that nanotheranostics will play an important role on predicting treatment responses, providing highly relevant insights for the improvement and understanding of targeted medicine, emphasizing the relevance of using both drugs and imaging agents within a single formulation. [2,3] In order to circumvent the limitation of the current medicines, several nanocarrier technologies are currently available or under development. [4] Drugs conjugated to synthetic polymers or serum proteins and drugs encapsulated in liposomes or other micro- or nanoparticles have been extensively explored and collectively termed polymer therapeutics. [5,6] The accumulation of these macromolecules in solid tumors due to a leaky capillary combined with a defective or absent lymphatic drainage system (enhanced permeation and retention effect, EPR effect) forms the rationale for developing polymer-based drug delivery systems. [7] Covalent linkage of a payload to a macromolecule leads to

40 41 42 43 44 45 46 47 48 49 50

R

N C O

38 39

U

36 37

R

30

34 35

18 19 20 21 22 23 24

C

28

32 33

F

R O

b

O

5 6 7

P

4

Harald R. Krüger a,1, Irene Schütz a,1, Aileen Justies a, Kai Licha c, Pia Welker c, Volker Haucke b, Marcelo Calderón a,2

D

Q23Q1

E

2

Imaging of doxorubicin release from theranostic macromolecular prodrugs via fluorescence resonance energy transfer

T

1

1 2

E-mail address: [email protected] (M. Calderón). These authors contributed equally. Fax: +49 30 838 52452.

the reduction of drug toxicity, elimination of undesirable side effects, and improvement of the solubility, stability, and prolonged blood halflife. [5,6,8] Such chemical linkages, however, can potentially induce steric hindrances and prevent interaction of the drug with its molecular target and thus render it inactive. Therefore, improved therapeutic efficacy can be realized when the active agent is linked to the carrier through a cleavable linker that is stable in blood and in healthy tissues but readily hydrolyzed upon entry into the target cancer cell or tumor. The over-expression of certain enzymes, an acidic and hypoxic environment in solid tumors, as well as targeting to the endolysosomal system offer several options for designing drug polymer conjugates that are preferentially cleaved within the tumor. [5,6,8–10] Doxorubicin (Dox) is a cytostatic agent that is currently used as a first line of treatment against several cancers/tumors. It has been used for more than 40 years as a free or liposomal formulation. [11] Great improvements by decreasing the non-specific toxicity and increasing the targeting of doxorubicin have been reported via the conjugation to various kinds of polymer nanoparticles as linear polymers, [12] dendritic polymers, [8,13,14] albumin, [15] bow-tie or star-like structures, [16] among others. Impressive results have been reported regarding the in vitro and in vivo efficacy of such polymer therapeutic approaches. However, little information is known about the intracellular drug release kinetics. Understanding the drug release mechanism and the intracellular fate of the polymer conjugates is crucial for the rational comprehending and improvement of the next generation of polymeric nanocarriers.

http://dx.doi.org/10.1016/j.jconrel.2014.08.018 0168-3659/© 2014 Published by Elsevier B.V.

Please cite this article as: H.R. Krüger, et al., Imaging of doxorubicin release from theranostic macromolecular prodrugs via fluorescence resonance energy transfer, J. Control. Release (2014), http://dx.doi.org/10.1016/j.jconrel.2014.08.018

51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76

2

89

2. Materials and methods

90

2.1. General methods

91

Chemicals, MPLC gradient solvents as well as deuterated solvents, and all reagents were used as purchased from commercial suppliers without any prior purification. Solvents were purified by conventional methods prior to use. All chemicals were of analytical grade and purchased from Fluka (Germany), Aldrich (Germany), and Merck (Germany), respectively. Polyglycerol (average MW 200 kDa, PDI = 1.6, Fig. S1) was prepared according to a slightly modified protocol in emulsion as described earlier. [20] Polyglycerol azide with 1% of the total hydroxyl groups bearing azido groups was prepared as previously described. [21] Briefly, polyglycerol azide was prepared by a two-step protocol starting from hyperbranched polyglycerol, conversion of OH groups into mesyl (Ms) groups followed by transformation of Ms groups into azide (N3) functionalities (Scheme S2). The indocarbocyanine IDCC dyes (Fig. S1) have been purchased from mivenion GmbH and were synthesized following literature procedures. [22,23] The (6-maleimidocaproyl) hydrazone derivative of doxorubicin (aldoxorubicin, Fig. S1) was prepared as described previously. [24] Water of Millipore quality (resistivity ~ 18 MΩ cm− 1, pH 5.6 ± 0.2) was used in all experiments and for preparation of all samples. All measurements were carried out with freshly prepared solutions at 25 °C. pH values were measured with a Scott instruments handylab pH meter at 25 °C. All reactions that involved air or water sensitive compounds were carried out in dried flasks under an argon atmosphere and dried solvents from the solvent purification system MB SPS 800, M. Braun Inertgas-Systeme GmbH, Garching, Germany. Column chromatography was conducted with RediSepRf Reversed-phase C18 Columns (average particle size: 40–63 μm, mesh: 230–400, average pore size: 60 Å) on a CombiFlashRf, Teledyne Isco, Inc., Lincoln, NE, USA. Thin layer chromatography (TLC) was conducted on Merck silica gel 60 F-254 and TLC silica gel 60 RP18 F-254s plates. Spots were visualized by UV light. 1H NMR and 13C spectra were recorded on a Jeol ECX-400 400 MHz spectrometer or a BrukerBioSpin (700 MHz) instrument at room temperature, and chemical shift values (δ) are given in ppm relative to internal standard MeOD-d4 (3.31 ppm). MS ESI-TOF analyses were performed on an Agilent 6210 ESI-TOF, Agilent Technologies, Santa Clara, CA, USA. The fluorescence spectra were recorded on a Jasco FP-6500 spectrofluorometer. UV/Vis spectra were recorded on a Scinco S-3100 spectrometer. Ultrafiltration was performed in solvent-resistant stirred cells from Millipore (Billerica, MA, USA) with Ultracel regenerated cellulose membranes (MWCO 5000 g mol− 1). CENTRIPREP-10concentrators from Amicon, FRG were used for ultracentrifugation. GPC analysis was performed by analytical size-exclusion HPLC using a Agilent-HPLC system with WinGPC Unity software from PSS; column: three 300 × 7.8 mm Polymer Laboratories PFgel mixed C; particle size: 5 μm; flow: 1.0 mL/min; isocratic; injection: 20 μL; mobile phase: water (0.05% NaN3); UV detection at 237 and 600 nm. Preparative column chromatography was performed on silica gel 60 (0.040–0.063 mm, 230–400 mesh ASTM). Detection was accomplished by UV irradiation (254 and 366 nm) or aqueous solutions of KMnO4.

102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139

143

Following the synthetic route described in Scheme 1, the theranostic macromolecular prodrug (TMP) was prepared in three consecutive steps: (a) synthesis of dye-labeled tri-functional linker, (b) coupling of the fluorescent linker to the polymer nanocarrier, and (c) coupling of the pH-sensitive Dox prodrug. As a highly orthogonal tri-functional linker, the di-protected fluorenylmethyloxycarbonyl (Fmoc) and tertbutyloxycarbonyl (Boc) protected amino acid lysine 1 was chosen. After aminolysis of propargyl amine and the carboxyl group of the lysine using benzotriazol-1-yl-oxy-tris-dimethylamino-phosphoniumhexafluorophosphate (BOP) and N,N′-diisopropylethylamine (DIPEA) with 95% yield, a Fmoc deprotection according to literature procedures was performed yielding 81% of product 2. [25] The IDCC dye was introduced via amide bond formation by an amino acid coupling strategy using N,N,N′,N′-Tetramethyl-O-(benzotriazol-1-yl)uroniumtetrafluoroborate (TBTU) and triethylamine (TEA) with 86% yield. Boc deprotection using trifluoroacetic acid (TFA) and slightly modified literature procedures gave 85% of product 3 (Scheme 1, a). [26] The IDCC dye-labeled linker derivatives were purified by reversed phase column chromatography and characterized by 1H-NMR spectroscopy (700 MHz), 13C-NMR spectroscopy (176 MHz), MS ESI-ToF, and UV-Vis. The next step was to conjugate the alkyne and dye containing linker construct 3 to the polymeric carrier PG (average MW 200 kDa) having a 1% azide functionalization by Huisgen 1,3-dipolar cycloaddition (Scheme 1b). The reaction was catalyzed by copper sulfate and sodium ascorbate as the reducing agent to form copper(I) in situ. [27] After 12 h of reaction in a mixture of water/MeOH (1/1 v/v) and DIPEA, product 4 was purified by ultrafiltration using water as solvent with one running-cycle with EDTA solution to remove all copper content yielding 84% of product. The dye conjugate 4 was characterized by UV-Vis spectroscopy, fluorescence spectroscopy, and gel permeation chromatography (GPC). The third and last step was the conjugation of the Dox prodrug aldoxorubicin via thiol-ene chemistry (Scheme 1c). Initial thiolation of the primary amine groups of 4 in situ was achieved by activation with 2iminothiolane in 50 mM PB solution (pH 7.4) for 20 min, followed by a selective Michael addition between the maleimide group of aldoxorubicin (Fig. S1, SI) and the sulfhydryl groups of the thiolated PG, yielding the TMP (5). The thiol group was added to the double bond of the maleimide group in a fast and selective reaction at room temperature forming a stable thioether bond. [14] After 2 h, the reaction solution was concentrated with a Centriprep® and purified by size exclusion chromatography (SEC) using Sephacryl S-100 gel. The conjugate was characterized by UV-Vis, GPC, and fluorescence spectroscopy. Non-cleavable and non-quenching conjugates were synthesized as control molecules 6 and 7 following a similar synthetic strategy (see details in SI section, Scheme S1). Molecule 6 bore Dox coupled via a more stable amide linker, whereas molecule 7 contained IDCC and aldoxorubicin randomly linked to the PG surface (Fig. 1).

144

2.3. Fluorescence analysis

191

The different conjugates 5, 6, and 7 (10 nM, concentration stated in Dox equivalents) were incubated in buffers of various pH (4, 5, 6, and 7.4) at 25 °C. For pH 7.4, 6, and 5 with 50 mM PB buffer and in the case of pH 4, 50 mM acetate buffer. The fluorescence was recorded exciting the samples at 500 nm, in the absorption range of the Dox, over time. The fluorescence emission peaked at 588 nm and increased over time. The lower the buffer pH, the faster the increase of the fluorescence intensity. The donor fluorescence was used to determine kinetic parameters. The concentration of TMP in the reaction mixture was determined by changes of the fluorescence at 588 nm.

192

O

R O

P

D

100 101

2.2. Synthesis of the theranostic macromolecular prodrug and controls

T

98 99

C

96 97

E

94 95

R

92 93

R

86 87

O

84 85

C

83

N

81 82

U

79 80

For SEC purification, SephadexTM G-25 superfine and SephacrylTMS- 140 100 HR were used. Human plasma was purchased from Merck KGaA, 141 Darmstadt. 142

F

88

Different attempts have been developed to monitor intracellular Dox release utilizing quantum dots, [17] fluorescent targeting probes, [18] or magnetic nanoparticles. [19] However, the development of a system that allows one to obtain spatial and temporal information about the cellular uptake and intracellular release kinetics of Dox from a polymer conjugate has not yet been reported. It is therefore necessary to develop functional probes that act as a reporter after Dox linkage and release from any kind of polymer nanoparticle. Such a universal approach would enable the evaluation of the great variety of polymer-Dox conjugates that have been described so far. We hypothesize that the native fluorescence of Dox could allow developing such a ubiquitous system using the fluorescence resonance energy transfer (FRET) phenomena.

E

77 78

H.R. Krüger et al. / Journal of Controlled Release xxx (2014) xxx–xxx

Please cite this article as: H.R. Krüger, et al., Imaging of doxorubicin release from theranostic macromolecular prodrugs via fluorescence resonance energy transfer, J. Control. Release (2014), http://dx.doi.org/10.1016/j.jconrel.2014.08.018

145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190

193 194 195 196 197 198 199 200 201

3

D

P

R O

O

F

H.R. Krüger et al. / Journal of Controlled Release xxx (2014) xxx–xxx

Q5

E

Scheme 1. A three-step strategy for the synthesis of TMP (5).

2.4. pH-dependent analysis

203 204

212 213

pH-dependent stability studies of the TMP (5)and the non-cleavable control 6 were carried out by HPLC at pH 4 (50 mM sodium acetate buffer) and pH 7.4 (50 mM sodium phosphate buffer). A 100 μM solution of each polymer conjugate (concentration stated in Dox equivalents) was incubated at room temperature in the respective buffer system and analyzed by analytical size-exclusion HPLC over 24 h using a Knauer-HPLC system with Geminyx System software; column: ProSEC 300s [300 × 7.8 mm], with a pre-column [80 × 7.8 mm] from Agilent, Germany; flow: 1.0 mL min-1, isocratic; injection volume: 20 μL; mobile phase: 30% acetonitrile/70% 10 mM sodium phosphate buffer, pH 7.4, detection at 495 nm.

214

2.5. Cell culture

215

HeLa cells were grown in low-glucose Dulbecco's modified Eagle's medium (DMEM; Lonza) supplemented with 10% (v/v) fetal bovine

216

C

E

R

210 211

R

208 209

N C O

206 207

serum (FBS), 1% glutamine, and 1% penicillin/streptomycin. Cells were 217 cultured at 37 °C and 5% CO2. 218 2.6. Fluorescence microscopy

219

Cells were seeded on MatTek glass bottom dishes (MatTek Corporation) and cultured overnight at 37 °C and 5% CO2, before live imaging cells were washed once with PBS and placed in a heating unit. Immediately after addition of imaging buffer (HBSS, 10 mM HEPES pH 7.4, 0.2% FBS) supplemented with 10 μM polymer conjugate (5, 6, or 7; concentration stated in Dox equivalents), images were acquired using the laser-scanning microscope LSM780 (Carl Zeiss MicroImaging GmbH, Jena, Germany) with a 63×/1.4 numerical aperture oilimmersion objective, λEx = 488 nm, detection window 508 –690 nm, pinhole 599 μm. Images were acquired for 60 min with a frame rate of 1 image every 10 min. After 120 min of conjugate addition, cells were washed with PBS and fixed with 4% paraformaldehyde/4% sucrose in PBS for 10 min at room temperature. Fixed samples were washed with PBS and subjected to imaging using the same setting as

220

U

205

T

202

Fig. 1. Schematic illustration of TMP (5) and the two controls, 6 (non-cleavable), and 7 (non-quenching). (For interpretation of the references to colour in this figure, the reader is referred to the web version of this article.)

Please cite this article as: H.R. Krüger, et al., Imaging of doxorubicin release from theranostic macromolecular prodrugs via fluorescence resonance energy transfer, J. Control. Release (2014), http://dx.doi.org/10.1016/j.jconrel.2014.08.018

221 222 223 224 225 226 227 228 229 230 231 232 233

4

247

2.7. Cytotoxicity assays

248

2.7.1. Cell viability and numbers HeLa cells were routinely cultured in RPMI medium, with 10% fetal calf serum (FCS), 2% glutamine, and penicillin/streptomycin (all from PAN Biotech) added. All cells were seeded into medium at 1 × 105 cells mL− 1, cultured at 37 °C with 5% CO2, and split 1:5 two times a week. Briefly, analyses were performed with cells cultured in 24-well plates. The 2 × 105 cells mL−1 were incubated in 1 mL culture medium containing increasing the concentration of test substance in 4 wells per concentration (concentrations ranged from 10−6 M to 10−9 M). After 2 days of culture, cell numbers and viability were analyzed in a cell counter and analyzer system. Experiments were done in quadruplicate. IC50 values and standard deviations were determined by plotting dose response curves using Microsoft Excel.

243 244

249 250 251 252 253 254 255 256 257 258 259 260 261 262

275

2.8. Plasma stability assay

276 277

286

Plasma stability of the TMP was assessed in vitro using the increase of the fluorescence of the released Dox as a test for the hydrolysis. Briefly, 10 μL (10 nM, concentration stated in Dox equivalents) of test substance was pipetted in a 96-well plates in 150 μL either normal human plasma, pH 7.4 (50 mM sodium acetate buffer), or pH 4 (50 mM sodium acetate buffer) as control. The fluorescence at a wavelength of 600 nm was read on a Microplate Spectrophotometer (Tecan, Infinite M200Pro) for 24 h at a temperature of 37 °C. Experiments were done in triplicate. Stability values were determined by plotting fluorescence intensity which is proportional to % degradation over time using Microsoft Excel (see Fig. S11, SI).

287

3. Results and discussion

288

3.1. Imaging of intracellular drug release with FRET

289 290

FRET is a non-radiative energy transfer process in which the energy is transferred from an excited state donor to a proximal ground state acceptor. The rate of energy transfer is highly dependent on many factors,

278 279 280 281 282 283 284 285

291

C

E

R

R

271 272

O

269 270

C

267 268

N

265 266

U

263 264

T

273 274

2.7.2. MTT assay Drug cytotoxicity was assessed in vitro using the MTT assay (cellular reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) as a test for metabolic activity of the cells. Briefly, 1 × 104 cells per well were plated in 96-well plates in 100 μL culture medium containing increasing the concentration of the test substance in 4 wells per concentration (concentrations ranged from 10−6 M to 10−9 M). After 3 days of culture, 10 μL MTT (5 mg mL−1 in PBS, obtained from Sigma Aldrich, Germany) was added to each well, and the plates were incubated for 4 h. The resulting formazan product was dissolved in acidic isopropanol, and the absorbance at a wavelength of 570 nm was read on a Microplate Spectrophotometer (Anthosht II, Microsystems). IC50 values and standard deviations were determined by plotting dose response curves using Microsoft Excel.

O

241 242

R O

240

P

238 239

D

236 237

such as the extent of spectral overlap, the relative orientation of the transition dipoles, and most importantly, the distance between the donor and acceptor molecules that should be lower than 10 nm. [28] Based on this, we designed a theranostic macromolecular prodrug (TMP) composed of (a) dendritic polyglycerol (PG) as polymeric nanocarrier, (b) Dox linked via a pH-sensitive hydrazone to (c) a trifunctional linker, and (d) an indodicarbocyanine dye (IDCC) attached in close proximity to Dox (Fig. 2a). The term theranostic is here utilized in the context of the definition described by Lammers et al., which describe those systems that provide insights into drug delivery with imaging approaches as nanotheranostics. [2]

F

245 246

stated above. Quantification analysis was performed using Fiji ImageJ software. HeLa cells were seeded on Ø 12 mm #1 glass coverslips (Thermo Scientific) and cultured overnight at 37 °C and 5% CO2. Cells were washed once with PBS and incubated for 2 h in DMEM (+10% FBS) supplemented with 10 μM TMP (stated in Dox equivalents). After two PBS washing steps, coverslips were fixed with 4% paraformaldehyde/4% sucrose in PBS and then mounted with Immu-Mount (Thermo Scientific). Images were acquired using the Nikon Eclipse Ti epifluorescence microscope with a 40×/1.3 numerical aperture oil-immersion objective, excitation filter BL HC 494/41, dichroic mirror HC BS 520, emission filter BL HC 582/75 (all from Semrock). Image processing was performed using micromanager.

E

234 235

H.R. Krüger et al. / Journal of Controlled Release xxx (2014) xxx–xxx

Fig. 2. (a) General structure of the TMP and disruption of the intramolecular energy transfer by cleavage of the hydrazone bond. (b) Proposed mode of action on cellular level. (For interpretation of the references to colour in this figure, the reader is referred to the web version of this article.)

Please cite this article as: H.R. Krüger, et al., Imaging of doxorubicin release from theranostic macromolecular prodrugs via fluorescence resonance energy transfer, J. Control. Release (2014), http://dx.doi.org/10.1016/j.jconrel.2014.08.018

292 293 294 295 296 297 298 299 300 301 302

H.R. Krüger et al. / Journal of Controlled Release xxx (2014) xxx–xxx

324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339

344 345 346 347 348 349 350 351 352

F

C

322 323

E

320 321

R

318 319

R

316 317

N C O

314 315

t1:1 t1:2

U

312 313

O

To find out whether the TMP (5) is capable of undergoing pHtriggered drug release and recovery of the Dox fluorescence, we analyzed the cleavage profile using GPC and fluorescence spectroscopy. TMP (5) and control samples (non-cleavable 6 and non-quenched 7, as well as conjugate 4, and a Polyglycerol-Dox conjugate) were incubated at different pH, mimicking the drop in pH during endocytosis and further endolysosomal trafficking in tumor cells. [2] Therefore, 10 nM solutions of the conjugates (concentration stated in Dox equivalents) in phosphate or acetate buffers of various pH (4, 5, 6, and 7.4) were prepared and analyzed. The fluorescence intensity (FI) was measured over

310 311

R O

343

309

P

3.2. Fluorescence analysis and GPC

307 308

D

342

305 306

time exciting the probes at 500 nm, in the absorption range of Dox. Due to the overlap of the emission spectra of Dox (λem = 580–620 nm) and the absorption spectra of IDCC (λex = 560–680 nm), and the distance between donor and acceptor being shorter than 10 nm, efficient FRET took place prior to the release of Dox from the TMP (5) (Fig. 3). We observed that following the hydrazone cleavage and Dox release at pH 4, 5, and 6, the fluorescence of Dox increased over time, as shown in Fig. 3a and b. On the other hand, the non-cleavable construct, conjugate 4 having only IDCC conjugated, and the Polyglycerol-Dox conjugate, yielded no measurable differences after incubation at acidic pH (Figs. S3 and S5, SI). Furthermore, the random non-quenching conjugate showed only marginal FRET efficiency, which further underscores the importance of the tri-functional linker that brings Dox and IDCC in close proximity (Fig. S4, SI). The quenching efficiency of the TMP (5) was calculated based on the equation S1 (SI) to be 94%, which is the highest value for a Dox-based system reported so far. It is worth to mention that the decrease of the induced acceptor fluorescence could not be followed over time due to a strong overlap with Dox fluorescence. From the release-profile studies shown in Fig. 3b, we determined that Dox release was both time and pH dependent, with almost no release under physiological conditions (pH 7.4). To visualize the regain of fluorescence upon cleavage of TMP (5), fluorescence images of the probe and control 6 were recorded exciting them at 450 nm (see Fig. 3c). Such results allowed us to determine kinetic parameters by following the donor fluorescence recovery over time (Fig. S6, SI). The kinetics of Dox release in the respective buffer solution was determined

T

340 341

PG was chosen as a biocompatible model nanocarrier (Fig. S1 in Supporting Information, SI), [29] but any other given polymer nanocarrier bearing an azide moiety could be used for this approach. Recently, it was demonstrated that chemically post-modified PG with a small content of azide groups presents sufficiently low zeta potentials, low interactions with serum albumin, and an enhanced cellular uptake by cancer cells, making it a good candidate for delivering anticancer agents. [30] It was found that PGs with molecular weights/sizes around 100–200 kDa/10 nm exhibit excellent cellular uptake and tumor accumulation, which make them excellent candidates for delivering diagnostic agents systemically. [31,32] We reported a proof of concept example that described the preparation of PG-Dox prodrugs that were flexible for drug loading by using an acid-sensitive hydrazone linker. The resulting polymer–drug conjugates showed optimal properties for in vitro and in vivo applications because of their high water solubility, an appropriate size for passive tumor toxicity, a high stability at physiological conditions, acid-sensitive properties, cellular internalization, and a favorable toxicity profile. PG-Dox conjugates with a high drug loading ratio showed significant improved antitumor efficacy over doxorubicin in an ovarian xenograft tumor model (A2780). [8] The IDCC dye (Fig. S1, SI) was selected because of its photophysical properties as a potential acceptor for Dox, e.g., degree of spectral overlap of Dox fluorescence and IDCC absorption (J), Förster distance (R0), and sufficient spatial separation of the emission wavelengths for accurate detection as shown in Table 1 and Fig. S2. Because of the near proximity of Dox and the IDCC dye (26 Å, calculated using equation S2), the emitted fluorescence of the drug upon excitation should be significantly decreased trough FRET. As a result, the pH-triggered cleavage of the hydrazone bond should release Dox and terminate the energy transfer. This allows monitoring the drug release by measuring the recovered Dox fluorescence emission. Facing cellular uptake and trafficking, the Dox fluorescence and cytotoxicity of the TMP will remain strongly decreased until cellular compartments with low pH values are reached (Fig. 2b). This theranostic approach would allow one to visualize and study Dox release and efficacy in live cells without invasive manipulations, in real time. The obtained information could provide crucial data regarding the therapeutic efficacy of conjugated drugs, their cell permeation efficiency, pathway, and mechanism of activation. [33–35]

E

303 304

5

Table 1 Photophysical parameters of donor-acceptor pair.

t1:3

Parameter

Value

t1:4 t1:5 t1:6 t1:7 t1:8 t1:9 t1:10

R0 (Å)a J (M−1 cm−1 nm4)b ΦDonorc ɛAcceptor (M−1 cm−1) λAbs. acceptor (nm) λEm. acceptor (nm) λEm. donor (nm)

41 7.68 × 1015 0.039 106 × 103 645 670 591

t1:11 t1:12 t1:13 t1:14

a

2

−4

1/6

Calculated using R0 = 0.2108 [k ΦDonor η J] . 4 Calculated using J= ∫ ∞ 0 FDonor(λ)εAcceptor(λ) λ dλ. c Determined on excitation at 480 nm using Ru(bpy)3Cl2 as reference (Φ = 0.028). [36]. b

Fig. 3. (a) Fluorescence emission spectra of the TMP (5) after incubation in acetate buffer (pH 4, 50 mM) at different time points. (b) Quantitative fluorescence analysis of the in vitro release of doxorubicin at 37 °C at different relevant pH. (c) Bright field (upper row) and fluorescence (lower row, λex = 450) pictures of the TMP (5) and noncleavable control 6 incubated in pH 4 and pH 7.4 for 8 h. (For interpretation of the references to colour in this figure, the reader is referred to the web version of this article.)

Please cite this article as: H.R. Krüger, et al., Imaging of doxorubicin release from theranostic macromolecular prodrugs via fluorescence resonance energy transfer, J. Control. Release (2014), http://dx.doi.org/10.1016/j.jconrel.2014.08.018

353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378

H.R. Krüger et al. / Journal of Controlled Release xxx (2014) xxx–xxx

The feasibility of using the TMP probe to study cellular uptake and endolysosomal drug release kinetics was evaluated by live cell imaging experiments in HeLa cells. To this aim, cells were incubated with 10 μM (concentration stated in Dox equivalents) of either one of the conjugates and a time lapse series was started immediately after conjugate addition for 60 min with a frame rate of 1 image every 10 min. The samples were excited at 488 nm, and Dox fluorescence was detected in the range from 512 nm to 684 nm. Exemplary fluorescence images of HeLa cells incubated with (5) or with the two control conjugates (6 and 7) are shown (Fig. 4a) at four different time points after addition. To address the question whether Dox fluorescence in the TMP probe is quenched via FRET, we analyzed the Dox sum fluorescence intensity for all conjugates using the same thresholds. The quantification of the 5 and 120 min time points is plotted in Fig. 4b. All determined intensity values were normalized to the non-cleavable control in each graph, respectively. Non-quenching control conjugate 7, in which the

E T C

403

E

401 402

R

399 400

R

397 398

O

395 396

C

393 394

N

391 392

U

389 390

F

388

385

O

3.3. Cellular analysis by life cell imaging

383 384

R O

387

381 382

distance between Dox and IDCC did not allow FRET, displayed high fluorescence intensity already 5 min post-addition to cells, which increased over time as more conjugate was internalized (Fig. 4a, b; compare 5 min and 120 min). The fluorescence sum intensity of the TMP probe appears elevated compared to the non-cleavable control. This effect becomes significant after prolonged cellular uptake for 120 min and likely reflects the cleavage of Dox from the prodrug, resulting in effective dequenching. These data indicate that the TMP probe undergoes low pHinduced cleavage when internalized into acidic endolysosomal compartments in living cells. For the quantification of the drug release and its subsequent accumulation in the cell nuclei after intracellular cleavage, we evaluated the Dox sum intensities using masks covering either the cytosol or the nuclear region. As seen in Fig. 4, c the nuclear-to-cytosol fluorescence ratio for the cleavable but non-quenching conjugate 7 did not change. Both values increased over time due to an increasing the concentration of polymer conjugate in the cell, increased accumulation of Dox in the nucleus, and absence of quenching of bound Dox. On the other hand, the nuclear-to-cytosol fluorescence ratio of internalized TMP (5) increases over time, reflecting the effective de-quenching and Dox accumulation in the cell nucleus. Moreover, there was also no change in the nuclear-to-cytosol fluorescence ratio of the quenched but noncleavable conjugate 6, because the missing cleavage of the drug did not allow a regaining of the fluorescence over time. These results suggest that following its internalization, TMP (5) undergoes rapid cleavage and free Dox quickly diffuses into the cell nucleus. The release of Dox from the conjugate most likely occurs in an acidic

D

386

by changes of the fluorescence at 588 nm giving a half-life of 107 min at pH 4 and a maximum drug release that was reached at 450 min (Fig. S6, inset). The cleavage profiles and kinetic parameters were consistent with the results from the GPC study (shown in Figs. S7 and S8, SI), which proves the correlation of fluorescence increase upon drug release. In addition, we have confirmed the stability of the TMP (5) in human plasma by measurement of the fluorescence signal over 24 h (see Fig. S11, SI).

379 380

P

6

Fig. 4. Life cell imaging in HeLa. (a) Light scanning microscopy images of Dox in HeLa cells incubated with TMP (5), non-cleavable control 6, and non-quenching control 7 at different time points. Images at 5, 30, and 60 min, respectively, were acquired during live cell imaging. Images at 120 min were taken after PFA fixation. Blow ups of indicated areas for TMP (5) and noncleavable control 6 at 60 min were 5% depth corrected to illustrate the distribution of Dox fluorescence either in the nucleus (N) or the cytosol (Cyt). Dashed lines indicate cell edges. Scale bar, 10 μm. (b) Quantification of Dox sum intensity for time points 5 min and 120 min. (c) Dox distribution in HeLa cells 5, 30, and 60 min after addition of TMP (5). Images were enhanced to indicate the accumulation of Dox fluorescence in the nucleus over time. Quantification on the right shows the ratio of nuclear/cytosolic intensity for all three conjugates (5, 6, and 7). Scale bars, 10 μm. (For interpretation of the references to colour in this figure, the reader is referred to the web version of this article.)

Please cite this article as: H.R. Krüger, et al., Imaging of doxorubicin release from theranostic macromolecular prodrugs via fluorescence resonance energy transfer, J. Control. Release (2014), http://dx.doi.org/10.1016/j.jconrel.2014.08.018

404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430

H.R. Krüger et al. / Journal of Controlled Release xxx (2014) xxx–xxx

458

4. Conclusion

459 460

480 481

In summary, we have introduced a novel approach to monitor the cellular uptake, intracellular release, and cytotoxicity of Dox from a TMP. We have designed a probe wherein the cytotoxicity is decreased and the fluorescence from Dox is quenched via FRET by attachment to a dye labeled dendritic polymer. Dox became fluorescent and cytotoxic upon cleavage of the hydrazone linkage to the polymer after endocytic cell uptake. The unprecedented decrease of 94% of the Dox fluorescence while conjugated to the polymer allowed quantification of the intracellular drug release and fast translocation to the cell nucleus. Moreover, the probe enabled study of the tracking of the nanocarrier by direct excitation of the acceptor dye. The chemical methodology could be used without any limitation for intracellular Dox fluorescence tracking for the broad variety of polymer-Dox conjugates reported so far. This highlights the versatility of the chemical methodology, which along with the ease and simplicity of the synthesis, might allow to quickly obtain important intracellular parameters leading towards an improvement of the performance of macromolecular-Dox prodrugs. Since neither cell disruption nor organelle isolation was needed, but rather the measurement could be done in real time, we postulate that such a FRET-based tool should be able to afford a more accurate assessment of the intracellular pathway from Dox and the nanocarriers, and the implications from the linker design on drug toxicity.

t2:1 t2:2

Table 2 Comparison of IC50 values of Dox and conjugates for HeLa cells.

453 454 455 456

461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479

t2:3

Compound

IC50 (nM) in HeLa cellsa

t2:4 t2:5 t2:6 t2:7

Dox PG-IDCC TMP (5) Non-cleavable control 6

2.6 ± 0.3 No Inhibitionb 112 ± 41 No inhibitionb

t2:8 t2:9 t2:10

487 488

Appendix A. Supplementary data

492

a Analyses of cell number after incubation with different concentrations of test substance for two days. b Maximum concentration used: 10−6 M.

489 490 491

Supplementary data to this article can be found online at http://dx. 493 doi.org/10.1016/j.jconrel.2014.08.018. 494 References

T

451 452

C

450

E

448 449

R

446 447

R

444 445

N C O

442 443

U

440 441

This study was supported by the NanoMatFutur grant from the BMBF, the German Funding Agency DFG (SFB 765/B4), and the Focus Area “Functional Materials at the Nanoscale” of the Freie Universität Berlin. We thank Prof. Dr. Rainer Haag and Dr. Felix Kratz for fruitful discussions and Dr. Pamela Winchester for proof reading.

F

457

It is worth mentioning that the cytotoxicity of Dox is exerted by its intercalation into cellular DNA resulting in the inhibition of transcription and protein biosynthesis. [38,39] To further correlate the changes in the fluorescence signal with the corresponding Dox release and translocation to the cell nucleus, we investigated the cytotoxicity of the TMP (5) and compared it to the non-cleavable construct 6. Because the amount of Dox released from the TMP influences the cell viability, we could correlate the change in fluorescence signal with the cytotoxicity of the samples. As expected, we observed that the TMP yielded a much higher cytotoxicity compared to the non-cleavable conjugate (see Table 2 and Table S1, as well as Fig. S10). As shown by our fluorescence imaging analysis, the non-cleavable control is unable to release Dox, and hence, free Dox cannot accumulate in the nucleus and intercalate into DNA. By contrast, the TMP was able to release the drug within the endolysosomal system, enabling its translocation and accumulation in the cell nucleus. These results are in agreement with previous reports in the literature that showed the lack of cytotoxicity from Dox linked through an amide linkage being unable to localize in the cell nucleus. [40]

486

484 485

O

439

Acknowledgments

R O

3.4. Cytotoxicity analysis

435 436

482 483

[1] J. Khandare, M. Calderón, N.M. Dagia, R. Haag, Multifunctional dendritic polymers in nanomedicine: opportunities and challenges, Chem. Soc. Rev. 41 (2012) 2824–2848. [2] T. Lammers, S. Aime, W.E. Hennink, G. Storm, F. Kiessling, Theranostic Nanomedicine, Acc. Chem. Res. 44 (2011) 1029–1038. [3] S. Svenson, in: R. K. (Ed.), Multifunctional Nanoparticles for Drug Delivery Applications, Imaging, Targeting, and Delivery, Springer New York, New York, 2012. [4] R. Duncan, M.J. Vicent, Polymer therapeutics-prospects for 21st century: the end of the beginning, Adv. Drug Delivery Rev. 65 (2013) 60–70. [5] R. Haag, F. Kratz, Polymere Therapeutika: Konzepte und Anwendungen, Angew. Chem. Int. Ed. 118 (2006) 1218–1237. [6] R. Haag, F. Kratz, Polymer therapeutics: concepts and applications, Angew. Chem. Int. Ed. Engl. 45 (2006) 1198–1215. [7] H. Maeda, G.Y. Bharate, J. Daruwalla, Polymeric drugs for efficient tumor-targeted drug delivery based on EPR-effect, Eur. J. Pharm. Biopharm. 71 (2009) 409–419. [8] M. Calderón, P. Welker, K. Licha, I. Fichtner, R. Graeser, R. Haag, F. Kratz, Development of efficient acid cleavable multifunctional prodrugs derived from dendritic polyglycerol with a poly(ethylene glycol) shell, J. Controlled Release 151 (2011) 295–301. [9] A. Godwin, K. Bolina, M. Clochard, E. Dinand, S. Rankin, S. Simic, S. Brocchini, New strategies for polymer development in pharmaceutical science—a short review, J. Pharm. Pharmacol. 53 (2001) 1175–1184. [10] F. Kratz, I.A. Müller, C. Ryppa, A. Warnecke, Prodrug Strategies in Anticancer Chemotherapy, ChemMedChem 3 (2008) 20–53. [11] Y. Barenholz, Doxil®—the first FDA-approved nano-drug: lessons learned, J. Controlled Release 160 (2012) 117–134. [12] T. Etrych, V. Šubr, J. Strohalm, M. Šírová, B. Říhová, K. Ulbrich, HPMA copolymerdoxorubicin conjugates: the effects of molecular weight and architecture on biodistribution and in vivo activity, J. Controlled Release 164 (2012) 346–354. [13] A.F. Hussain, H.R. Krüger, F. Kampmeier, T. Weissbach, K. Licha, F. Kratz, R. Haag, M. Calderón, S. Barth, Targeted delivery of dendritic polyglycerol–doxorubicin conjugates by scFv-SNAP fusion protein suppresses EGFR + cancer cell growth, Biomacromolecules 14 (2013) 2510–2520. [14] M. Calderón, R. Graeser, F. Kratz, R. Haag, Development of enzymatically cleavable prodrugs derived from dendritic polyglycerol, Bioorg. Med. Chem. Lett. 19 (2009) 3725–3728. [15] F. Kratz, S. Azab, R. Zeisig, I. Fichtner, A. Warnecke, Evaluation of combination therapy schedules of doxorubicin and an acid-sensitive albumin-binding prodrug of doxorubicin in the MIA PaCa-2 pancreatic xenograft model, Int. J. Pharm. 441 (2013) 499–506. [16] C.C. Lee, E.R. Gillies, M.E. Fox, S.J. Guillaudeu, J.M. Frechet, E.E. Dy, F.C. Szoka, A single dose of doxorubicin-functionalized bow-tie dendrimer cures mice bearing C-26 colon carcinomas, Proc. Natl. Acad. Sci. U. S. A. 103 (2006) 16649–16654. [17] V. Bagalkot, L. Zhang, E. Levy-Nissenbaum, S. Jon, P.W. Kantoff, R. Langer, O.C. Farokhzad, Quantum dot—aptamer conjugates for synchronous cancer imaging, therapy, and sensing of drug delivery based on bi-fluorescence resonance energy transfer, Nano Lett. 7 (2007) 3065–3070. [18] S. Santra, C. Kaittanis, O.J. Santiesteban, J.M. Perez, Cell-specific, activatable, and theranostic prodrug for dual-targeted cancer imaging and therapy, J. Am. Chem. Soc. 133 (2011) 16680–16688. [19] S.S. Banerjee, D.-H. Chen, Multifunctional pH-sensitive magnetic nanoparticles for simultaneous imaging, sensing and targeted intracellular anticancer drug delivery, Nanotechnology 19 (2008) 505104. [20] R.K. Kainthan, E.B. Muliawan, S.G. Hatzikiriakos, D.E. Brooks, Synthesis, characterization, and viscoelastic properties of high molecular weight hyperbranched polyglycerols, Macromolecules 39 (2006) 7708–7717. [21] S. Roller, H. Zhou, R. Haag, High-loading polyglycerol supported reagents for Mitsunobu- and acylation-reactions and other useful polyglycerol derivatives, Mol. Divers. 9 (2005) 305–316. [22] J. Pauli, K. Licha, J. Berkemeyer, M. Grabolle, M. Spieles, N. Wegner, P. Welker, U. Resch-Genger, New fluorescent labels with tunable hydrophilicity for the rational

P

438

433 434

Furthermore, the TMP could easily be adapted to incorporate targeting ligands such as folates, peptides, antibodies, etc. The simplicity of the synthetic strategy described makes this approach attractive for developing and optimizing efficient anticancer drug delivery systems.

E

437

environment inside endosomes and lysosomes post-internalization by endocytosis. This hypothesis is supported by our previous cellular uptake studies of PGs with MW of 200 kDa, which have provided evidence for the endocytic uptake of such polymers. [30,31,37] Additionally, the IDCC label allowed us to track the conjugate independently and determine its intracellular fate. As expected, we observed that the polymer remained in the cytosol after drug cleavage (see Fig. S9, SI).

D

431 432

7

Please cite this article as: H.R. Krüger, et al., Imaging of doxorubicin release from theranostic macromolecular prodrugs via fluorescence resonance energy transfer, J. Control. Release (2014), http://dx.doi.org/10.1016/j.jconrel.2014.08.018

495 496 497 498 499 500 Q4 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555

[25] [26]

[27] [28]

[29] [30]

N

C

O

R

R

E

C

T

E

D

P

[31]

F

[24]

[32] H.-Y.H.H.R. Krüger, D. Vats, M. Vizovisek, D. Buhlmann, J. Rajkovic, K.U. Wendt, A. Duro-Castano, M.J. Vicent, M. Calderón, M. Rudin, O. Plettenburg, B. Turk, C. Schultz, R. Haag, In vivo Imaging of Cathepsin B Activity in Tumor Microenvironment with Novel Polymeric Smart Probe, 10th International Symposium on Polymer Therapeutics: from Laboratory to Clinical Practice, Valencia, Spain, 2014. [33] O. Redy, D. Shabat, Modular theranostic prodrug based on a FRET-activated selfimmolative linker, J. Controlled Release 164 (2012) 276–282. [34] H. Komatsu, Y. Shindo, K. Oka, J.P. Hill, K. Ariga, Ubiquinone-rhodol (UQ-Rh) for fluorescence imaging of NAD(P)H through intracellular activation, Angew. Chem. Int. Ed. 53 (2014) 3993–3995. [35] M. Akamatsu, H. Komatsu, T. Mori, E. Adams, R. Shin, H. Sakai, M. Abe, J.P. Hill, K. Ariga, Intracellular imaging of cesium distribution in arabidopsis using cesium green, ACS Appl. Mater. Interfaces 6 (2014) 8208–8211. [36] R. Anand, S. Ottani, F. Manoli, I. Manet, S. Monti, A close-up on doxorubicin binding to [gamma]-cyclodextrin: an elucidating spectroscopic, photophysical and conformational study, RSC Adv. 2 (2012) 2346–2357. [37] M. Calderón, S. Reichert, P. Welker, K. Licha, F. Kratz, R. Haag, Receptor mediated cellular uptake of low molecular weight dendritic polyglycerols, J. Biomed. Nanotechnol. 10 (2014) 92–99. [38] N. Altan, Y. Chen, M. Schindler, S.M. Simon, Defective acidification in human breast tumor cells and implications for chemotherapy, J. Exp. Med. 187 (1998) 1583–1598. [39] P.-S. Lai, P.-J. Lou, C.-L. Peng, C.-L. Pai, W.-N. Yen, M.-Y. Huang, T.-H. Young, M.-J. Shieh, Doxorubicin delivery by polyamidoamine dendrimer conjugation and photochemical internalization for cancer therapy, J. Controlled Release 122 (2007) 39–46. [40] H.S. Yoo, T.G. Park, Biodegradable polymeric micelles composed of doxorubicin conjugated PLGA–PEG block copolymer, J. Controlled Release 70 (2001) 63–70.

O

[23]

design of bright optical probes for molecular imaging, Bioconjug. Chem. 24 (2013) 1174–1185. K. Licha, C. Hessenius, A. Becker, P. Henklein, M. Bauer, S. Wisniewski, B. Wiedenmann, W. Semmler, Synthesis, characterization, and biological properties of cyanine-labeled somatostatin analogues as receptor-targeted fluorescent probes, Bioconjug. Chem. 12 (2001) 44–50. F. Kratz, A. Warnecke, K. Scheuermann, C. Stockmar, J. Schwab, P. Lazar, P. Drückes, N. Esser, J. Drevs, D. Rognan, C. Bissantz, C. Hinderling, G. Folkers, I. Fichtner, C. Unger, Probing the cysteine-34 position of endogenous serum albumin with thiolbinding doxorubicin derivatives. Improved efficacy of an acid-sensitive doxorubicin derivative with specific albumin-binding properties compared to that of the parent compound, J. Med. Chem. 45 (2002) 5523–5533. F. Kratz, U. Beyer, M.T. Schutte, Drug-polymer conjugates containing acid-cleavable bonds, Crit. Rev. Ther. Drug Carrier Syst. 16 (1999) 245–288. M.C. Bröhmer, S. Mundinger, S. Bräse, W. Bannwarth, Chelating carboxylic acid amides as robust relay protecting groups of carboxylic acids and their cleavage under mild conditions, Angew. Chem. Int. Ed. Engl. 50 (2011) 6175–6177. R.A. Evans, The rise of azide–alkyne 1,3-dipolar ‘click’ cycloaddition and its application to polymer science and surface modification, Aust. J. Chem. 60 (2007) 384–395. K.E. Sapsford, L. Berti, I.L. Medintz, Materials for fluorescence resonance energy transfer analysis: beyond traditional donor–acceptor combinations, Angew. Chem. Int. Ed. Engl. 45 (2006) 4562–4589. M. Calderón, M.A. Quadir, S.K. Sharma, R. Haag, Dendritic polyglycerols for biomedical applications, Adv. Mater. 22 (2010) 190–218. J. Khandare, A. Mohr, M. Calderón, P. Welker, K. Licha, R. Haag, Structurebiocompatibility relationship of dendritic polyglycerol derivatives, Biomaterials 31 (2010) 4268–4277. S. Reichert, P. Welker, M. Calderón, J. Khandare, D. Mangoldt, K. Licha, R.K. Kainthan, D.E. Brooks, R. Haag, Size-dependant cellular uptake of dendritic polyglycerol, Small 7 (2011) 820–829.

U

556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 612

H.R. Krüger et al. / Journal of Controlled Release xxx (2014) xxx–xxx

R O

8

Please cite this article as: H.R. Krüger, et al., Imaging of doxorubicin release from theranostic macromolecular prodrugs via fluorescence resonance energy transfer, J. Control. Release (2014), http://dx.doi.org/10.1016/j.jconrel.2014.08.018

586 587 588 589 590 591 592 593 594 595 596 597 598 599 600 601 602 603 604 605 606 607 608 609 610 611

Imaging of doxorubicin release from theranostic macromolecular prodrugs via fluorescence resonance energy transfer.

Herein we present a FRET-based theranostic macromolecular prodrug (TMP) composed of (a) dendritic polyglycerol (PG) as polymeric nanocarrier, (b) doxo...
2MB Sizes 0 Downloads 5 Views