Multifunctional Polymeric Prodrug with Simultaneous Conjugating

Subscriber access provided by AUT Library

Biological and Medical Applications of Materials and Interfaces

Multifunctional Polymeric Prodrug with Simultaneous Conjugating Camptothecin and Doxorubicin for pH/Reduction Dual-Responsive Drug Delivery Shuxiang Dong, Yue Sun, Jie Liu, Lei Li, Jinlin He, Mingzu Zhang, and Peihong Ni ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 29 Jan 2019 Downloaded from http://pubs.acs.org on January 29, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 10 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Materials & Interfaces

Multifunctional Polymeric Prodrug with Simultaneous Conjugating Camptothecin and Doxorubicin for pH/Reduction DualResponsive Drug Delivery Shuxiang Dong, Yue Sun, Jie Liu, Lei Li, Jinlin He, Mingzu Zhang, and Peihong Ni* College of Chemistry, Chemical Engineering and Materials Science, State and Local Joint Engineering Laboratory for Novel Functional Polymeric Materials, Jiangsu Key Laboratory of Advanced Functional Polymer Design and Application, Suzhou Key Laboratory of Macromolecular Design and Precision Synthesis, Soochow University, Suzhou 215123, P. R. China ABSTRACT: Amphiphilic polymeric prodrugs show improved therapeutic indices with respect to traditional hydrophobic anticancer

drugs because these prodrugs can self-assemble into nanoparticles, prolong the circulation of drugs in the blood, improve the accumulation of drugs in the disease site, reduce the side effects of drugs and achieve therapeutic effect. Here we describe a novel pH/reduction dual-responsive polymeric prodrug, abbreviated as CPT-sspoly(BYP-hyd-DOX-co-EEP), with simultaneous conjugating camptothecin (CPT) and doxorubicin (DOX), wherein BYP and EEP represent two cyclic phosphate monomers, respectively, that is, 2-(but-3-yn-1-yloxy)-2-oxo-1,3,2-dioxaphospholane and 2-ethoxy-2-oxo-1,3,2-dioxaphospholane. This prodrug was prepared through a polyphosphoester-DOX conjugate using a CPT derivative (CPT-ss-OH) as the initiator. Camptothecin is linked to the terminal of polyphosphoester via disulfide carbonate, which is easy to break up under intracellular reductive environment and release the parent CPT; while DOX was efficiently incorporated onto the pendants of polyphosphoester through hydrazone bond (-hyd-), which would be cleaved in the intracellular acidic medium. We show that the stable nanoparticles formed by the prodrug could release CPT and DOX simultaneously in the tumor cell environment. The results of MTT assay demonstrate that the prodrug, which binds two antitumor drugs simultaneouly, has the properties of low toxicity, dual pH/reductionsensitiveness, biocompatibility, biodegradability and effective tumor therapy. KEYWORDS:

pH/Reduction dual-responsiveness, Amphiphilic prodrug, Polyphosphoester, CuAAC “click” reaction

■ INTRODUCTION In recent years, the incidence of malignant tumors has become a public health problem, and even a social problem that must be attached great importance to. More than 10 million new cancer cases and more than 7 million deaths are reported worldwide every year.1 So far, more than 400 different chemotherapeutic drugs have been found, which can be targeted at specific cancer treatment.2 Among them, doxorubicin (DOX) and camptothecin (CPT) are the two most commonly used antineoplastic drugs. DOX can inhibit the synthesis of RNA and DNA. It has the strongest inhibitory effect on RNA. Therefore, it has a wide antitumor spectrum and kills many kinds of cancer cells.3-5 CPT can selectively inhibit topoisomerase I (Topo I) and bind to the complex formed by Topo I-DNA to stabilize the complex, so that the broken DNA chain cannot be reconnected, thus preventing DNA replication and RNA synthesis. It can inhibit many kinds of tumors and has no cross-resistance with commonly used anticancer drugs.6-8 However, due to their hydrophobicity, toxicity, poor solubility, non-specific distribution and side effects, the clinical efficacy of these two antineoplastic drugs is limited.9-12

For improving the above shortcomings and increasing the effect of drug chemotherapy, an effective way is to adopt nanotechnology, i.e. physical adsorption and encapsulation, or chemical conjugation between the carriers and drugs. Nanocarriers can be inorganic or polymeric nanosized materials (diameter 1–100 nm). Because of their high surface area-volume ratio, they can carry different hydrophobic drugs into the body. Functional nanomaterials, especially stimulus-responsive nanomaterials, play an important role in drug delivery. They can prevent premature drug leakage and side effects in normal tissues.13,14 Extracellular and intracellular stimuli of tumors include pH, glutathione (GSH), light and heat, etc. Water-soluble polymers and small molecular drugs can form amphiphilic polymer prodrugs by stimulusresponsive group as the linker. It can effectively improve the hydrophobicity of drugs and form polymeric prodrug nanoparticles. In external stimulus environments, these sensitive linking groups are destroyed and the drug is released.15-20 In a typical prodrug system, in contrast to the physical encapsulation of drugs into nanoparticles, the drugs are covalently linked to a polymer to become temporarily dormant. The inactive polymeric prodrug is further changed into an active drug in vivo for therapeutic under

1 ACS Paragon Plus Environment

ACS Applied Materials & Interfaces 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

the triggering of some specific stimuli. In this field, many efforts have focused on biocompatible and biodegradable polymers as delivery systems21,22 that are responsive to intracellular changes of stimuli including pH gradient,23 redox species,24 photoirradiation,25 enzymes,26 as well as magnetic and electric ﬁelds.27 The high concentration gradient of GSH between the intracellular (∼10 mM) and extracellular environment (∼2 µM) can be used as an ideal in vivo trigger to design redox responsive polymer prodrugs or micelles.28,29

Page 2 of 10

microenvironment and acidic condition, respectively, to allow CPT and DOX parent drugs release. ■ Results and Discussion Polymer Synthesis and Structural Characterizations.

The relative experimental details are described in the Supporting Information. Four steps were adopted to synthesize the pH/reduction dual-responsive polymeric prodrug CPT-sspoly(BYP-hyd-DOX-co-EEP), as shown in Scheme 1. (1) Synthesis of disulfide carbonate-containing initiator CPT-ss-OH according to the previous literature;49 (2) Preparation of hydrazone-containing DOX-hyd-N3 by adopting a previously published procedure;6 (3) Preparation of polyphosphoester-based random copolymer CPTss-poly(BYP-co-EEP) containing pendant alkynyl groups by onepot ROP reaction of BYP and EOP initiated by the hydroxyl group of CPT-ss-OH; (4) Preparation of the final dualresponsive polymeric prodrug via the CuAAC “click” reaction between DOX-hyd-N3 and CPT-ss-poly(BYP-co-EEP). The disulfide carbonate and hydrazone groups in the polymeric prodrug can be cleaved under the intracellularly reductive and acidic medium milieu. The two anticancer drugs had been conjugated onto one macromolecule and afforded a high loading content, which had a capability of self-assembly in aqueous solution.

Among the reported biocompatible and biodegradable polymers, polyphosphoesters (PPEs) is worthy of attention and is expected to be widely used in the field of biomedicine.30-34 Leong,35 Wooley,36 Fréchet,37 Yan31 and other groups have proved this point. In our previous research, we designed several stimuliresponsive polymeric prodrugs. Using various synthetic methods, we prepared polyphosphate-based prodrugs, which were linked with doxorubicin (DOX), camptothecin (CPT) and paclitaxel (PTX), respectively. Also, we demonstrated their controlled drug delivery and biomedical applications.38-40 Polyphosphoester-based nanocarriers with defined hydrophilicity can control the stealth properties of the polymer shell and that the logP value of the copolymer controls the composition of the protein corona and the cell interaction.41 However, there are few reports on the simultaneous bonding of two types of antineoplastic drugs on a polyphosphate molecule, in which the drugs are linked by acidsensitive and/or reduction-sensitive groups. Herein, we present a novel pH/reduction dual-responsive polymeric prodrug CPT-ss-poly(BYP-hyd-DOX-co-EEP), which was prepared by random ring-opening polymerization (ROP) of cyclic phosphate monomers, using a CPT derivative (CPT-ss-OH) containing S-S bond and hydroxyl group as an initiator. At first, the two monomers were 2-(but-3-yn-1-yloxy)-2-oxo-1,3,2dioxaphospholane (BYP) and 2-ethoxy-2-oxo-1,3,2dioxaphospholane (EOP), respectively. The resulting product was a CPT-terminated random copolyphosphoester CPT-sspoly(BYP-co-EEP), in which the disulfide carbonate linkage had reductive property so that it would be broken in the presence of glutathione and CPT is efficiently released.42-44 Second, the pendant alkynyl groups in this random copolymer was further bonded an azide and hydrazone-functionalized DOX derivative (DOXhyd-N3) through a highly efficient CuAAC “click” reaction,45-47 leading to the successful synthesis of pH/reduction dualresponsive polymeric prodrug CPT-ss-poly(BYP-hyd-DOX-co-EEP). The acid-sensitive hydrazone linker could be cleavable and release DOX either in the slightly acidic of extracellular environment of tumor cells or in the acidic intracellular environment of endosomal (pH ∼5.5−6.0) and lysosomal (pH ∼4.5−5.0) compartments; Thirdly, the polyphosphoester (PPE)-based copolymers have good biocompatibility and biodegradability, and their degradation products and time can be adjusted by means of precise chemistry.48

Figure 1. 1H NMR spectra of (A) CPT-ss-OH and (B) CPT-sspoly(BYP-co-EEP) in CDCl3.

The resulting CPT-ss-poly(BYP-hyd-DOX-co-EEP) is an amphiphilic polymeric prodrug, which simultaneously conjugate two types of anticancer drugs, that is, CPT and DOX. This prodrug can self-assemble into nanoparticle in aqueous solution. Phosphodiesterase I in vivo can accelerate the degradation of the main chain of polyphosphate and dissociate the prodrug nanoparticles.30,31 The disulfide carbonate linkage and acid-sensitive hydrazone bond would be rapidly cleaved in the presence of a reductive


Page 3 of 10 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Scheme 1. Synthetic routes of (1) azide-terminated hydrazone-containing DOX derivative DOX-hyd-N3, (2) disulfide carbonate-containing initiator CPT-ss-OH, (3) reduction and pH dual-sensitive prodrug CPT-ss-poly(BYP-hyd-DOX-co-EEP) via the ROP and CuAAC “click” reactions, and (4) mechanism of CPT release from CPT-ss-poly(BYP-hyd-DOX-co-EEP) in the presence of GSH.

tion time of CPT, CPT-ss-OH and CPT-ss-poly(BYP-co-EEP), there is a shift in elution time of CPT, indicating the structural changes. All these results confirmed CPT was successfully conjugated to CPT-ss-poly(BYP-co-EEP). Table 1 summarizes the chemical compositions and molecular weights of the random copolymer CPT-ss-poly(BYP-co-EEP) including calculated data from 1H NMR results and GPC data. According to the 1H NMR spectrum in Figure 1(B), the molecular weight of CPT-ss-poly(BYP-co-EEP) was calculated by Eqn. (1):

All samples were characterized by 1H NMR, GPC, FT-IR, HPLC and LC/MS analysis, respectively, to determine their chemical structure and molecular weights. The 1H NMR spectra in Figure 1 show the characteristic chemical shifts from the initiator CPT-ssOH and the random copolymer CPT-ss-poly(BYP-co-EEP). From these 1H NMR spectra, we can find all of the chemical shifts corresponding to the protons of the polymer and prodrug. Figure 1(A) shows the 1H NMR spectrum of CPT-ss-OH. Figure 1(B) is the 1H NMR spectrum of CPT-ss-poly(BYP-co-EEP) initiated by CPT-ssOH. With comparison of the two spectra, the new proton signals, which appeared at δ 1.33 ppm (peak 7), δ 2.58 ppm (peak 4), δ 4.14 ppm (peak 3 and 6), and δ 4.25 ppm (peak 1 and 2) in the Figure 1(B), can be ascribed to the protons of CPT-ss-poly(BYP-co-EEP). We also utilized HPLC analysis to obtain the different elution time of the samples. As shown in Figure 2, with a comparison of the elu-

M n,NMR =


A4 2 Ad

× 176.1 +

A7 3 Ad

× 152.1 + 528.6

(1)


Page 4 of 10

Moreover, the test data from LC/MS analysis is m/z 697.3 Da, which is nearly identical with the calculated value 696.7 Da (Figure S2).

where A4 and Ad are the integral values of peak 4 at δ 2.58 ppm and peak d at δ 8.38 ppm in Figure 1(B); A7 represents the integral value of peak 7 at δ 1.33 ppm in Figure 1(B); 176.1 and 152.1 are the molar mass of one repeat unit of PBYP and PEEP, respectively; 528.6 is the molar mass of the CPT molecule. The number-average molecular weights and molecular weight distributions (PDIs) of CPT-ss-poly(BYP-co-EEP) were measured by GPC analysis which are shown in Figure S3. It can be found that the molecular weights of the polyphosphoesters determined by 1H NMR and GPC analysis seem to have a difference, which may be ascribed to the different structures of these polymers and GPC measurement only provide a relative value based on polystyrene standards.38,50,51

Figure 3. FT-IR spectra of (A) DOX, (B) DOX-hyd-N3.

Figure 2. HPLC curves of (A) CPT, (B) CPT-ss-OH, and (C) CPT-sspoly(BYP-co-EEP) Table 1 Characterization data of the chemical compositions and molecular weights of random copolymer CPT-ss-poly(BYP-co-EEP). Sample

M n, NMR -1 a)

(g mol )

M n, GPC (g mol-1) b)

PDI b)

CPT-ss-poly(BYP28-coEEP33)

10400

4500

1.34


17900

5400

1.39


20500

3600

1.23

Figure 4. HPLC curves of (A) CPT-ss-poly(BYP-hyd-DOX-co-EEP), (B) DOX-hyd-N3, and (C) DOX.

Finally, the pH/reduction dual-responsive polymeric prodrug CPT-ss-poly(BYP-hyd-DOX-co-EEP) was obtained via the CuAAC “click” reaction between the azide group of DOX-hyd-N3 and alkynyl group of CPT-ss-poly(BYP-co-EEP). Figure 5 shows the 1H NMR spectrum of CPT-ss-poly(BYP30-hyd-DOX-co-EEP80), from which all the chemical shifts assigned to the protons of the polymeric prodrug could be observed. Typically, the appearance of the characteristic chemical shifts at δ 7.92 ppm and δ 11.4 ppm, which were ascribed to DOX, demonstrated the successful modification of CPT-ss-poly(BYP-co-EEP). HPLC measurement was also utilized to further confirm the structure. As shown in Figure 4, the elution time for DOX-hyd-N3 has shifted from 3.1 min to 1.8 min, proving the successful synthesis of CPT-ss-poly(BYP-hyd-DOX-co-EEP).

a)

Calculated from 1H NMR spectra. b) Determined by GPC with DMF as the solvent and polystyrene as standards.

The functional hydrazone-containing DOX-hyd-N3 was synthesized by using previously published procedures,51 and its chemical structure was proved by 1H NMR, FT-IR, HPLC and LC/MS analysis. The 1H NMR spectrum DOX-hyd-N3 is shown in Figure S1. FT-IR, HPLC and LC/MS were also utilized to confirm the successful preparation of DOX-hyd-N3. Figure 3 shows the FT-IR spectra of the original DOX and DOX-hyd-N3. The absorption peak of azide group at 2097 cm-1 in Figure 3(B) indicates that the azide group has been successfully connected into DOX. In addition, the results from HPLC analysis shown in Figure 4 demonstrate that the elution time for DOX and DOX-hyd-N3 were 5.2 min and 3.1 min, respectively, indicating that the chemical structure has changed.


Page 5 of 10 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Materials & Interfaces shape and well dispersed in aqueous solution. The corresponding size distribution curve of the nanoparticles measured by DLS displayed unimodal distribution as shown in Figure 7(B). There is a little difference in the results of particle size measure by TEM (~69 nm) and DLS (~90 nm), which might be due to the collapse of hydrophilic chains in TEM samples during drying.

Figure 7. (A) TEM image of the CPT-ss-poly(BYP-hyd-DOX-co-EEP) nanoparticles (scale bar = 200 nm); (B) the particles size distribution curve corresponding to the TEM sample with the concentration of 1.0 mg mL-1.

Figure 5. 1H NMR spectrum of CPT-ss-poly(BYP30-hyd-DOX-co-EEP80) in DMSO-d6. Self-Assembly of the Polymeric Prodrug

The reduction and pH dual-responsive properties have been given after introducing the disulfide carbonate and hydrazone linkers. For investigating the pH/reduction dual-responsive degradation of CPT-ss-poly(BYP-hyd-DOX-co-EEP) nanoparticles, the DLS analysis was used to monitor the size change under different pH values or GSH conditions at different time intervals. We selected three conditions to study the size PDI changes of the prodrug nanoparicles as follows: (A) PB 7.4; (B) PB 5.0, and (C) PB 7.4+10 mM GSH. In absence of GSH, there is no obvious change over 24 h as shown in Figure 8(A). Under the acidic condition, that is, at PB 5.0 (Figure 8(B)), the particle size increased from 90 nm to 570 nm at 2 h interval and larger nanoparticles with an average particle size of >1000 nm were formed over 24 h. In contrast, an obvious size change for CPT-ss-poly(BYP-hyd-DOX-co-EEP) nanoparticles was observed in the presence of 10 mM GSH as shown in Figure 8(C), suggesting the good reduction-triggered degradability. Within 2 h, the average particle size increased from 90 nm to 680 nm with the increasing size PDIs from 0.184 to 0.722. The large nanoparticles with an average diameter of >1000 nm were formed after 24 h. It could deduce that the size increase due to the cleavage of the disulfide linker in the presence of GSH or/and the hydrazone linker in the acidic condition. When CPT or DOX drugs were cleaved from the terminal group and side chain of CPT-ss-poly(BYP-hyd-DOX-coEEP), the amphiphilicity of nanoparticles could be changed, which led to loose structure and ultimately would further form large aggregates due to the inferior stability.

Figure 6. Intensity of the fluorescence emission spectrum of pyrene as a function of logarithm concentration of the CPT-ss-poly(BYP-hyd-DOX-coEEP) prodrug in aqueous solution.

In order to evaluate the self-assembly behavior of this polymeric prodrug, the critical aggregation concentration (CAC) value of CPT-ss-poly(BYP-hyd-DOX-co-EEP) was determined by a fluorescence probe method using pyrene as the hydrophobic probe. In theory, when the concentration of the amphiphilic prodrug up to a certain value in aqueous solution, it can self-assemble into nanoparticles (NPs) with hydrophobic PBYP segment containing DOX and CPT as the core and the hydrophilic PEEP parts as the shell. As shown in Figure 6, the CAC value was determined to be 43 mg L-1. Considering particle size ( D z ) and particle size distribution (size PDI) are important parameters for the drug delivery nanocarriers, the dynamic light scattering (DLS) and transmission electron microscope (TEM) analyses were utilized to investigate the selfassembly behavior of the polymeric prodrug. Figure 7(A) showed the TEM image of the CPT-ss-poly(BYP-hyd-DOX-co-EEP) NPs, from which we can find that these nanoparticles mainly formed spherical



Page 6 of 10

Figure 8. Particle size distribution histograms of CPT-ss-poly(BYP-hyd-DOX-co-EEP) nanoparticles at various time intervals under different conditions: (A) PB 7.4, (B) pH 5.0, and (C) PB 7.4 + 10 mM GSH.

release from the polymeric prodrug nanoparticles were evaluated in various media: (I) pH 7.4, (II) pH 7.4 with 2 µM GSH, (III) pH 7.4 with 10 mM GSH, and (IV) pH 5.0. The DOX content was determined to be about 8.0% by fluorescence spectrophotometer. As shown in Figure 9(A), in the absence of GSH or in the presence of 2 µM GSH, only small amount of CPT release (~20%) after incubating for 80 h, which exhibited minimal drug leakage under the physiological condition. While in the presence of 10 mM GSH, the CPT release was significantly accelerated within the same period of time, and approximately 47% of CPT was released from the nanoparticles

In Vitro Drug Release

It has been reported that the tumor extracellular microenvironments (pH 6.5-7.2) and endosomes/lysosomes (pH 4.5-6.5) are more acidic than normal tissue microenvironments (pH ~7.4), and the intracellular GSH level (2-10 mM) is several orders of magnitude higher than the extracellular GSH level (2-10 µM).52-54 To investigate the reduction and pH-responsive release behavior of CPT-ss-poly(BYP-hyd-DOX-co-EEP) nanoparticles, in vitro drug


Page 7 of 10 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


after incubation for 80 h. These results suggested the favorable GSH-induced reduction-responsive drug release behavior. Moreover, as shown in Figure 9(B), a similar accelerated DOX release behavior could also be observed in the low pH condition with ~61% of DOX released after 100 h incubation, while only about 17% of DOX was released at pH 7.4 at the same time, indicating the acidresponsive drug release behavior. The reasons for these phenomena can be explained as follows: high GSH concentration or the acidic environment could cause the cleavage of the disulfide bonds or hydrazone linkers, which resulted in the loose structure of nanoparticles and accelerated release of original CPT or DOX drugs. All these results indicated that the simultaneous release of DOX and CPT could be achieved via the GSH or acid-triggered disassembly of prodrug nanoparticles.

Figure 10. Cell viability of L929, HepG2 and HeLa cells treated with poly(BYP-co-EEP) at different concentrations for 48 h incubation.

Figure 11. Cell viability of (A), (B) HeLa cells and (C), (D) HepG2 cells, which were treated with CPT-ss-poly(BYP-hyd-DOX-co-EEP) nanoparticles and free CPT or DOX with different drug dosages for 48 h incubation. The IC50 value of CPT-ss-poly(BYP-hyd-DOX-co-EEP) was calculated by GraphPad Prism 5 software.

Figure 9. In vitro drug release profiles of (A) CPT from CPT-sspoly(BYP-hyd-DOX-co-EEP) nanoparticles in PB (pH 7.4) with different GSH concentrations; (B) DOX from CPT-ss-poly(BYP-hyd-DOX-co-EEP) nanoparticles in PB (pH 7.4) or acetate buffer (pH 5.0) at 37 °C.

We studied the effects of the two free drugs and the polymeric prodrug on the different cancer cells. Figure 11(A) and (B) show the cell viability of HeLa celles in the case of CPT and DOX, respectively. Figure 11(C) and (D) depict the cell viability of HepG2 cells in the case of CPT and DOX, respectively. In either free drug or prodrug system, the cell viability decreased with the increase of drug concentrations and showed a dose-dependent anti-proliferative activity. Furthermore, the prodrug nanoparticles showed a higher half maximal inhibitory concentration (IC50) values compared with that of the free CPT and DOX. The IC50 values of free CPT, free DOX and prodrug for HeLa cells are about 0.48 mg L-1, 1.83 mg L-1, and 4.04 mg L-1, respectively. For HepG2 cells, the IC50 values of free CPT, free DOX and prodrug are about 9.51 mg L-1, 6.88 mg L-1,

In Vitro Cytotoxicity

Generally speaking, biocompatibility is an important point for the application of polymer materials in drug delivery. For proving the biocompatibility of the polymer backbone, the cell viability of poly(BYP-co-EEP) without CPT conjugation against L929, HepG2 and HeLa cells was evaluated by MTT assays, respectively. Figure 10 shows that all the cell viability exceeded 80% with polymer concentrations up to 0.2 mg mL−1, indicating the low cytotoxicity of this polymer backbone.



and 6.75 mg L-1, respectively. They might be attributed to the decrease of cytotoxicity influenced by the poly(BYP-co-EEP) and would not impair normal cell before reaching cancer cell. In addition, it took time for the prodrug to release the parent drug in comparison to free CPT or DOX. Overall, the in vitro cytotoxicity results indicate that CPT-ss-poly(BYP-hyd-DOX-co-EEP) nanoparticles possess a good antitumor activity.

Page 8 of 10

■ CONCLUSIONS We report a new polymeric prodrug, which was conjugated with two types of anticancer drugs, CPT and DOX. A camptothecin derivative (CPT-ss-OH) was first used to initiate the one-pot random ring-opening polymerization of two kinds of phosphoester monomers. And then a doxrobicin derivative DOX-hyd-N3 was introduced onto the side chain of polyphosphoester via CuAAC click chemistry to yield a novel pH/reduction dual-responsive polymeric prodrug CPT-ss-poly(BYP-hyd-DOX-co-EEP). In the in vitro drug release results, approximate 47% of CPT and 61% of DOX released simultaneously from the prodrug nanoparticles under tumor intracellular condition. All the cell viability exceeded 80% with different concentrations of polyphosphoester poly(BYP-co-EEP) against L929, HeLa, and HepG2 cells, respectively. The prodrug nanoparticles could efficiently inhibit the tumor cell proliferation. Meanwhile, the prodrug nanoparticles could be effectively uptaken into HeLa cells by endocytosis. Therefore, these biodegradable dual-responsive prodrug nanoparticles could be considered as potential drug carriers for cancer therapy.

Cellular Uptake

The cellular uptake and intracellular drug release behavior of CPT-ss-poly(BYP-hyd-DOX-co-EEP) nanoparticles against HeLa cells were real-time monitored by live cell imaging system. As shown in Figure 12(A), DOX fluorescence was observed in the cytoplasm of HeLa cells after incubation with prodrug nanoparticles for 1 h, indicating the successful internalization of CPT-ss-poly(BYP-hyd-DOX-coEEP) nanoparticles and efficient release of DOX in HeLa cells. Furthermore, with the increase of incubation time from 1 h to 5 h, a substantial enhancement of the intracellular DOX fluorescence was observed. In comparison, free DOX showed relatively lower fluorescence intensity in HeLa cells within the same incubation time as shown in Figure 12(B), which was due to the different cellular internalization mechanisms between the prodrug nanoparticles (endocytosis) and free drug (diffusion). These results imply that the prodrug nanoparticles could be internalized by HeLa cells, and the drug could be efficiently released from the prodrug nanoparticles.

■ ASSOCIATED CONTENT Supporting Information

The Supporting Information is available free of charge on the ACS Publications website at DOI: Experimental details and characterization data. Synthesis of CPT-ssOH, DOX-hyd-N3, and Polymer CPT-ss-poly(BYP-co-EEP), Polymeric Prodrug CPT-ss-poly(BYP-hyd-DOX-co-EEP); 1H NMR spectrum of DOX-hyd-N3; LC-MS spectrum of DOX-hyd-N3 ； GPC traces of CPT-ss-poly(BYP-co-EEP). (PDF) ■ AUTHOR INFORMATION Corresponding Author * Tel: +86 512 65882047; E-mail: [email protected] ORCID Peihong Ni: 0000-0003-4572-3213 Jinlin He: 0000-0003-3533-2905

Notes

The authors declare no competing financial interest. ■ ACKNOWLEDGMENTS

The authors are grateful for the financial supports from the National Natural Science Foundation of China (21374066), the Major Program of the Natural Science Project of Jiangsu Higher Education Institutions (15KJA150007), Natural Science Foundation of Jiangsu Province (BK20171212), a project funded by the Priority Academic Program Development (PAPD) of Jiangsu Higher Education Institutions, and the Foundation of Key Laboratory of Synthetic and Biological Colloids, Ministry of Education, Jiangnan University (No. JDSJ2017-05). We are also grateful to Professor Jian Liu (FUNSOM, Soochow University) for his valuable help in the cellrelated tests.

Figure 12. Fluorescence images of HeLa cells incubated with (A) CPTss-poly(BYP-hyd-DOX-co-EEP) nanoparticles for different incubation times and (B) free DOX. The DOX dosage was 0.5 mg L-1. For each panel, images from left to right show cell nuclei stained by H 33342 (blue), DOX (red) and overlays of the blue and red images. The scale bars correspond to 50 µm in all images.


Page 9 of 10 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Materials & Interfaces Chem. 2014, 5, 1529-1544. (19) Nicolas, J. Drug-Initiated Synthesis of Polymer Prodrugs: Combining Simplicity and Efficacy in Drug Delivery. Chem. Mater. 2016, 28, 1591-1606. (20) Cheetham, A. G.; Chakroun, R. W.; Ma, W.; Cui, H. SelfAssembling Prodrugs. Chem. Soc. Rev. 2017, 46, 6638-6663. (21) Li, C. M.; Madsen, J.; Armes, S. P.; Lewis, A. L. A New Class of Biochemically Degradable, Stimulus-Responsive Triblock Copolymer Gelators. Angew. Chem., Int. Ed. 2006, 45, 3510-3513. (22) Ding, M. M.; Zeng, X. Z.; He, X. L.; Li, J. H.; Tan, H., Fu, Q. Cell Internalizable and Intracellularly Degradable Cationic Polyurethane Micelles as a Potential Platform for Efficient Imaging and Drug Delivery. Biomacromolecules 2014, 15, 2896-2906. (23) Wang, L.; Liu, G. H.; Wang, X R.; Hu, J. M.; Zhang, G. Y.; Liu, S. Y. Acid-Disintegratable Polymersomes of pHResponsive Amphiphilic Diblock Copolymers for Intracellular Drug Delivery. Macromolecules 2015, 48, 7262-7272. (24) Lale ,S. V.; Kumar, A.; Prasad, S.; Bharti, A. C.; Koul, V. Folic Acid and Trastuzumab Functionalized Redox Responsive Polymersomes for Intracellular Doxorubicin Delivery in Breast Cancer. Biomacromolecules 2015, 16, 1736-1752. (25) Zhou, Y.; Ye, H.; Chen, Y. B.; Zhu R. Y.; Yin, L. C. Photoresponsive Drug/Gene Delivery Systems. Biomacromolecules 2018, 19, 1840-1857. (26) Hu, J. M.; Zhang, G. Q.; Liu, S. Y. Enzyme-Responsive Polymeric Assemblies, Nanoparticles and Hydrogels. Chem. Soc. Rev. 2012, 41, 5933-5949. (27) Krack, M.; Hohenberg, H.; Kornowski, A.; Lindner, P.; Weller, H.; Forster, S. Nanoparticle-Loaded Magnetophoretic Vesicles. J. Am. Chem. Soc. 2008, 130, 7315-7320. (28) Mura, S.; Nicolas, J.; Couvreur, P. Stimuli-Responsive Nanocarriers for Drug Delivery. Nat. Mater. 2013, 12, 9911003. (29) Dai, L. L.; Li, J. H.; Zhang, B. L.; Liu, J. J.; Luo, Z.; Cai, K. Y. Redox-Responsive Nanocarrier Based on Heparin EndCapped Mesoporous Silica Nanoparticles for Targeted Tumor Therapy in Vitro and in Vivo. Langmuir 2014, 30, 7867-7877. (30) Bauer, K. N.; Tee, H. T.; Velencoso, M. M.; Wurm, F. R. Main-Chain Poly(phosphoester)s: History, Syntheses, Degradation, Bio-and Flame-Retardant Applications. Prog. Polym. Sci. 2017, 73, 61-122. (31) Liu, J. Y.; Huang, W.; Pang, Y.; Yan, D. Y. Hyperbranched Polyphosphates: Synthesis, Functionalization and Biomedical Applications. Chem. Soc. Rev. 2015, 44, 3942-3953. (32) Zhang, F.; Khan, S.; Li, R.; Smolen, J. A.; Zhang, S.; Zhu, G.; Su, L.; Jahnke, A. A.; Elsabahy, M.; Chen, X.; Wooley, K. L. Design and Development of Multifunctional Polyphosphoester-Based Nanoparticles for Ultrahigh Paclitaxel Dual Loading. Nanoscale 2017, 9, 15773-15777. (33) Boyer, C.; Teo, J.; Phillips, P.; Erlich, R. B.; Sagnella, S.; Sharbeen, G.; Dwarte, T.; Duong, H. T. T.; Goldstein, D.; Davis, T. P.; Kavallaris, M.; McCarroll, J. Effective Delivery of siRNA into Cancer Cells and Tumors using WellDefined Biodegradable Cationic Star Polymers. Mol. Pharmaceutics 2013, 10, 2435-2444. (34) Tsao, Y. Y. T.; Wooley, K. L. Synthetic, Functional Thymidine-Derived Polydeoxyribonucleotide Analogues from a Six-Membered Cyclic Phosphoester. J. Am. Chem. Soc. 2017, 139, 5467-5473. (35) Wang, J.; Mao, H. Q.; Leong, K. W. A Novel Biodegradable Gene Carrier Based on Polyphosphoester. J. Am. Chem. Soc. 2001, 123, 9480-9481.

■ REFERENCES (1)

(2) (3)

(4)

(5)

(6)

(7)

(8)

(9)

(10) (11)

(12)

(13)

(14)

(15)

(16) (17)

(18)

Bray, F.; Ferlay, J.; Laversanne, M.; Brewster, D. H.; Mbalawa, C. G.; Kohler, B.; Piñeros, M.; Steliarova-Foucher, E.; Swaminathan, R.; Antoni, S.; Soerjomataram, I.; Forman, D. Cancer Incidence in Five Continents: Inclusion Criteria, Highlights from Volume X and the Global Status of Cancer Registration. Int. J. Cancer 2015, 137, 2060-2071. Penny, L. K.; Wallace, H. M. The Challenges for Cancer Chemoprevention. Chem. Soc. Rev. 2015, 44, 8836-8847. Agudelo, D.; Bourassa, P.; Berube, G.; Tajmir-Riahi, H. A. Review on the Binding of Anticancer Drug Doxorubicin with DNA and tRNA: Structural Models and Antitumor Activity. J. Photochem. Photobiol. B 2016, 158, 274-279. Chen, C.; Lu, L.; Yan, S.; Yi, H.; Yao, H.; Wu, D.; He, G.; Tao, X.; Deng, X. Autophagy and Doxorubicin Resistance in Cancer. Anti Canc. Drugs 2018, 29, 1-9. Meredith, A. M.; Dass, C. R. Increasing Role of the Cancer Chemotherapeutic Doxorubicin in Cellular Metabolism. J. Pharm. Pharmacol. 2016, 68, 729-741. Wall, M. E.; Wani, M. C.; Cook, C. E.; Palmer, K. H.; McPhail, A .T.; Sim, G. A. Plant antitumor agents. I. The Isolation and Structure of Camptothecin, a Novel Alkaloidal Leukemia and Tumor Inhibitor from Camptotheca Acuminate. J. Am. Chem. Soc. 1966, 88, 3888-3890. Thomas, C. J.; Rahier N. J.; Hecht S. M. Camptothecin: Current Perspectives. Bioorg. Med. Chem. 2004, 12, 15851604. Krishnan, P.; Rajan, M.; Kumari, S.; Sakinah, S.; Priya, S. P.; Amira, F.; Danjuma, L.; Ling, M. P.; Fakurazi, S., Arulselvan, P.; Higuchi, A.; Arumugam, R.; Alarfaj, A. A.; Munusamy, M. A.; Hamat, R. A.; Benelli, G.; Murugan, K.; Kumar, S. S. Camptothecin with β-cyclodextrin-EDTAFe3O4 Nanoparticle Conjugated Nanocarriers as an Anticolon Cancer (HT29) Drug. Sci. Rep. 2017, 7, 10962. Chen, X. J.; Parelkar, S. S.; Henchey, E.; Schneider, S., Emrick, T. PolyMPC-Doxorubicin Prodrugs. Bioconjugate Chem 2012, 23, 1753-1763. Kim, B. Y.S.; Rutka, J. T.; Chan, W. C. W. Nanomedicine. New Engl. J Med., 2010, 363, 2434-2443. Shen, Y. Q.; Jin, E. L.; Zhang, B.; Murphy, C. J.; Sui, M. H.; Zhao, J.; Wang, J. Q.; Tang, J. B.; Fan, M. H.; Kirk, E. V., Murdoch, W. J. Prodrugs Forming High Drug Loading Multifunctional Nanocapsules for Intracellular Cancer Drug Delivery. J. Am. Chem. Soc. 2010, 132, 4259-4265. Shi, J.; Kantoff, P. W.; Wooster, R.; Farokhzad, O. C. Cancer Nanomedicine: Progress, Challenges and Opportunities. Nat. Rev. Cancer 2017, 17, 20-37. Peer, D.; Karp, J. M.; Hong, S.; Farokhzad, O. C.; Margalit, R.; Langer, R. Nanocarriers as an Emerging Platform for Cancer Therapy. Nat. Nanotechnol. 2007, 2, 751-760. Fan, W.; Yung, B.; Huang, P.; Chen, X. Y. Nanotechnology for Multimodal Synergistic Cancer Therapy. Chem. Rev. 2017, 117, 13566-13638. Tao, X. Y.; Jia, N.; Cheng, N. H.; Ren, Y. H.; Cao, X. N.; Liu, M.; Wei, D. Z.; Wang, F. Q. Design and Evaluation of a Phospholipase D Based Drug Delivery Strategy of Novel Phosphatidyl-Prodrug. Biomaterials 2017, 131, 1-14. Khandare, J.; Minko, T. Polymer-Drug Conjugates: Progress in Polymeric Prodrugs. Prog. Polym. Sci. 2006, 31, 359-397. Kratz, F.; Müller, I. A.; Ryppa, C.; Warnecke, A. Prodrug Strategies in Anticancer Chemotherapy. Chem. Med. Chem. 2008, 3, 20-53. Delplace, V.; Couvreur, P. ; Nicolas, J. Recent Trends in the Design of Anticancer Polymer Prodrug Nanocarriers. Polym.



Page 10 of 10

(45) Mohapatra, H.; Ayarza, J.; Sanders, E. C.; Scheuermann, A. M.; Griffin, P. J.; Esser-Kahn, A. P. Ultrasound Promoted Step-Growth Polymerization and Polymer Crosslinking Via Copper Catalyzed Azide-Alkyne “Click” Reaction. Angew. Chem., Int. Ed. 2018, 57, 1-6. (46) Huang, Z.; Zhou, Y.; Wang, Z.; Li, Y.; Zhang, W.; Zhou, N.; Zhang, Z.; Zhu, X. Recent Advances of CuAAC Click Reaction in Building Cyclic Polymer. Chin. J. Polym. Sci. 2017, 35, 317-341 (47) Corrigan, N.; Shanmugam, S.; Xu, J.; Boyer, C. Photocatalysis in Organic and Polymer Synthesis. Chem. Soc. Rev. 2016, 45, 6165-6212. (48) Schöttler, S.; Becker, G.; Winzen, S.; Steinbach, T.; Mohr, K.; Landfester, K.; Mailänder, V.; Wurm, F. R. Protein Adsorption is Required for Stealth Effect of Poly(ethylene glycol)- and Poly(phosphoester)-Coated Nanocarriers. Nat. Nanotechnol. 2016, 11, 372-377. (49) He, D.; Zhang, W.; Deng, H.; Huo, S.; Wang, Y. F.; Gong, N.; Deng, L.; Liang, X.; Dong, A. J. Self-Assembling Nanowires of an Amphiphilic Camptothecin Prodrug Derived from Homologous Derivative Conjugation. Chem. Commun. 2016, 52, 14145-14148. (50) Zhai, X.; Huang, W.; Liu, J. Y.; Pang, Y.; Zhu, X. Y.; Zhou, Y. F.; Yan, D. Y. Micelles from Amphiphilic Block Copolyphosphates for Drug Delivery. Macromol. Biosci. 2011, 11, 1603-1610. (51) He, J. L.; Zhang, M. Z.; Ni, P. H. Rapidly in Situ Forming Polyphosphoester-Based Hydrogels for Injectable Drug Delivery Carriers. Soft Matter 2012, 8, 6033-6038. (52) Wang, Y., Luo, Q. J.; Zhu, W. P.; Li, X. D.; Shen, Z. Q. Reduction/pH Dual-Responsive Nano-Prodrug Micelles for Controlled Drug Delivery. Polym. Chem. 2016, 7, 26652673. (53) Chiang, Y. T.; Yen, Y. W.; Lo, C. L. Reactive Oxygen Species and Glutathione Dual Redox-Responsive Micelles for Selective Cytotoxicity of Cancer. Biomaterials 2015, 61, 150-161. (54) Hu, Y. W.; Du, Y. Z.; Liu, N.; Liu, X.; Meng, T. T.; Cheng, B. L.; He, J. B.; You, J.; Yuan, H.; Hu, F. Q. Selective Redox-Responsive Drug Release in Tumor Cells Mediated by Chitosan Based Glycolipid-Like Nanocarrier. J. Control. Release 2015, 206, 91-100.

(36) Zhang, F.; Zhang, S.; Pollack, S. F.; Li, R.; Gonzalez, A. M.; Fan, J.; Zou, J.; Leininger, S. E.; Sanders, A. P.; Johnson, R.; Nelson, L. D.; Raymond, J. E.; Elsabahy, M.; Hughes, D. M. P.; Lenox, M. W.; Gustafson, T. P.; Wooley, K. L. Improving Paclitaxel Delivery: In Vitro and In Vivo Characterization of PEGylated Polyphosphoester-Based Nanocarriers. J. Am. Chem. Soc. 2015, 137, 2056-2066. (37) Lee, C. C.; Gillies, E. R.; Fox, M. E.; Guillaudeu, S. J.; Fréchet, J. M. J.; Dy, E. E.; Szoka, F. C. A Single Dose of Dxorubicin-Functionalized Bow-tie Dendrimer Cures Mice Bearing C-26 Colon Carcinomas. Proc. Natl. Acad. Sci. U. S. A 2006, 103, 16649-16654. (38) Zhang, Q. Q.; He, J. L.; Zhang, M. Z.; Ni, P. H. A Polyphosphoester-Conjugated Camptothecin Prodrug with Disulfide Linkage for Potent Reduction-Triggered Drug Delivery. J. Mater. Chem. B 2015, 3, 4922-4932. (39) Du, X. Q.; Sun, Y.; Zhang, M. Z.; He, J. L.; Ni, P. H. PolyphosphoesterCamptothecin Prodrug with ReductionResponse Prepared Via Michael Addition Polymerization and Click Reaction. ACS Appl. Mater. Interfaces 2017, 9, 13939-13949. (40) Cao, Y. W.; He, J. L.; Liu, J.; Zhang, M. Z.; Ni, P. H. Folate-Conjugated Polyphosphoester with Reversible CrossLinkage and Reduction Sensitivity for Drug Delivery. ACS Appl. Mater. Interfaces 2018, 10, 7811-7820. (41) Simon, J.; Wolf, T.; Klein, K.; Landfester, K.; Wurm, F. R.; Mailänder, V. Hydrophilicity Regulates the Stealth Properties of Polyphosphoester-Coated Nanocarriers. Angew. Chem., Int. Ed. 2018, 57, 5548-5553. (42) Hu, X. L.; Hu, J. M.; Tian, J.; Ge, Z. S.; Zhang, G. Y.; Luo, K. F.; Liu, S. Y. Polyprodrug Amphiphiles: Hierarchical Assemblies for Shape- Regulated Cellular Internalization, Trafficking, and Drug Delivery. J. Am. Chem. Soc. 2013, 135, 17617-17629. (43) Lee, M. H.; Kim, J. Y.; Han, J. H.; Bhuniya, S.; Sessler, J. L.; Kang, C.; Kim, J. S. Direct Fluorescence Monitoring of the Delivery and Cellular Uptake of a Cancer-Targeted RGD Peptide-Appended Naphthalimide Theragnostic Prodrug. J. Am. Chem. Soc. 2012, 134, 12668-12674. (44) Cai, K.; He, X.; Song, Z.; Yin, Q.; Zhang, Y.; Uckun, F. M.; Jiang, C.; Cheng, J. Dimeric Drug Polymeric Nanoparticles with Exceptionally High Drug Loading and Quantitative Loading Efficiency. J. Am. Chem. Soc., 2015, 137, 34583461.


Multifunctional Polymeric Prodrug with Simultaneous Conjugating

Recommend Documents