MethotrexateâCamptothecin Prodrug Nanoassemblies as a Versatile

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Methotrexate-Camptothecin Prodrug Nanoassemblies as A Versatile Nanoplatform for Biomodal Imaging-Guided Self-Active Targeted and Synergistic Chemotherapy Yang Li, Jinyan Lin, Jinyuan Ma, Liang Song, Huirong Lin, Bowen Tang, Dengyue Chen, Guanghao Su, Shefang Ye, Xuan Zhu, Fang-Hong Luo, and Zhenqing Hou ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b10027 • Publication Date (Web): 18 Sep 2017 Downloaded from http://pubs.acs.org on September 19, 2017

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ACS Applied Materials & Interfaces

MethotrexateMethotrexate-Camptothecin Prodrug Nanoassemblies as A Versatile Nanoplatform for Biomodal Imagingmaging-Guided Selfelf-Active Targeted and Synergistic Synergistic Chemotherapy Yang Li, a‡ Jinyan Lin, a, e‡ Jinyuan Ma, b Liang Song, a Huirong Lin, d Bowen Tang, b, f Dengyue Chen, b Guanghao Su, g Shefang Ye, a Xuan Zhu, b Fanghong Luo, c* Zhenqing Hou, a*

a

Key Laboratory of Biomedical Engineering of Fujian Province & Research

Center of Biomedical Engineering of Xiamen, Department of Biomaterials, College of Materials, Xiamen University, Xiamen 361005, P. R. China b

School of Pharmaceutical Science, Fujian Provincial Key Laboratory of

Innovative Drug Target Research, Xiamen University, Xiamen 361005, P. R. China c

Cancer Research Center, Medical College, Xiamen University, Xiamen 361005, P.

R. China d

State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics &

Center for Molecular Imaging and Translational Medicine, School of Public Health, Xiamen University, Xiamen 361005, P. R. China e

College of Chemistry and Chemical Engineering, State Key Laboratory of

Physical Chemistry of Solid Surfaces, Xiamen University, Xiamen 361005, P. R. China f

College of Pharmacy, Western University of Health Science, Pomona, California

91766, USA g

Children’s Hospital of Soochow University, Suzhou 215025, P. R. China

‡ Dr.

Y. Li and J. Lin contributed equally to this work.

Corresponding author: Zhenqing Hou. E-mail address: [email protected] Corresponding author: Fanghong Luo. E-mail address: [email protected]. 1 ACS Paragon Plus Environment


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ABSTRACT: BSTRACT: “All-in-one” carrier-free-based nano-multi-drug self-delivery system could combine triple advantages of small molecules, nanoscale characteristics, and synergistic combination therapy together. Researches have showed that dual-acting small molecular methotrexate (MTX) could target and kill the folate receptor-overexpressing cancer cells. Inspired by this mechanism, a novel collaborative “early-phase tumor-selective targeting and late-phase synergistic anticancer” approach was developed for the self-assembly of chemotherapeutic drug-drug conjugate, which showed various advantages of more simplicity, efficiency, and flexibility over the conventional approach that was only based on single or combination cancer chemotherapy. MTX and 10-hydroxyl camptothecin (CPT) were chosen to conjugate through ester linkage. Because of the amphiphilicity and ionicity, MTX-CPT conjugates as molecular building blocks could self-assemble into MTX-CPT nanoparticles (MTX-CPT NPs) in aqueous solution, thus notably improving the aqueous solubility of CPT and the membrane permeability of MTX. The MTX-CPT NPs with a precise drug-to-drug ratio showed pH-/esterase-responsive drug release, sequential function “Targeting to Anticancer” switch, and real-time monitoring fluorescence “Off-On” switch. By doping with a lipophilic near-infrared (NIR) cyanine dye (e.g., 1’-dioctadecyl-3, 3, 3’, 3’-tetramethylindotricarbocyanine iodide, DiR), the prepared DiR-loaded MTX-CPT NPs acted as an effective probe for in vivo NIR fluorescence (NIRF) and photoacoustic (PA) dual-modal imaging. Both in vitro and in vivo studies demonstrated that MTX-CPT NPs could specifically co-deliver multi-drug to different sites of action with distinct anticancer mechanisms to kill folate receptor-overexpressing tumor cells in a synergistic way. This novel, simple, and highly convergent self-targeting nano-multi-drug co-delivery system exhibited great potential in cancer therapy.

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KEYWORDS: EYWORDS: self-assembly, small molecular nanodrug, self-targeted drug delivery, combination cancer therapy, real-time monitoring and imaging



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1. INTRODUCTION Currently chemotherapy is still considered one of the most widely used modalities for cancer therapy because of its high efficiency. Although a large number of chemotherapeutic drugs have been utilized extensively in clinical practice, the traditional single-drug chemotherapy often suffered from poor therapeutic efficacy and severe drug resistance after frequent administration due to the complexity and heterogeneity of various tumors. 1 Thus, combination chemotherapy which uses multi-drug with different sites and mechanisms of action at the maximum tolerated dose has been considered as an alternative strategy to overcoming the limitations of single-drug chemotherapy. 2 However, due to the small molecular size and unique pharmacokinetics of individual chemotherapeutic

drug,

the

clinical

trials

of

traditional

combination

chemotherapy via cocktail administration still face enormous challenges including rapid blood/renal clearance, nonspecific biodistribution, low accumulation in tumor tissues, serious toxicity to normal tissues, and limited synergistic efficacy, etc. 1-3 In recent years, nanotechnology has emerged as an innovative and promising strategy to circumvent the drawbacks of traditional combination chemotherapy. 1, 4

Rapid development in nanocarriers-based delivery systems including

liposomes, polymeric nanoparticles (NPs), dendrimers, micelles, protein NPs, and inorganic NPs have offered unparalleled opportunities in delivering multiple chemotherapeutic drugs with different pharmacokinetic and pharmacodynamic mechanisms via enhanced permeability and retention (EPR) effect.

1, 5-6

Compared to the delivery of a single chemotherapeutic drug, the co-delivery of multiple chemotherapeutic drugs by nanocarriers to the same tumor cells has shown synergistic therapeutic efficacy and inhibition of drug resistance in vitro and in vivo.

7-8

Over the past decade, significant progress has been made in the

rapid development of sophisticated, carrier-based, and targeting nanoscale 4 ACS Paragon Plus Environment

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multi-drug delivery systems for improving therapeutic efficiency.

9-10

However,

their clinical translations are still far below the public expectation due to several major drawbacks, for instance, the low drug loading capacity (typically less than 10%),

the

inert

carrier

materials-related

degradation/metabolism/excretion concern,

11-13

toxicity

issues,

the

and even the complexity of

designing a multicomponent nanomedicine (containing at least the carrier and two or more anticancer drugs, and often tumor-targeting ligands, and/or excipients).

14-15

Moreover, only a few selected carriers have received the

approval from US Food and Drug Adminstration (FDA). 16-17 Instead of using any carrier material, amphiphilic small molecular prodrugs have drawn many pharmacists’ attention due to their simple structure and convenient assembly.

13-14

Compared to the traditional nanocarrier-based drug

delivery system, the carrier-free self-delivery nanodrug system could not only avoid the concern of significant toxicity of drug carriers but also achieve both nanoscale advantages and quantitative/high drug payload simultaneously. 18-22

15,

However, to date, the EPR effect could only enhance the passive

accumulation of nanodrug at tumor sites, the poor tumor site-specific delivery and lack of active cellular internalization still hampered the efficacy of cancer chemotherapy.

5-6

Thus, the drug-drug conjugate-based nanoassemblies using

ligand targeting strategy might be provided with superiorities of active targeting, prodrug and carrier-free nanodrug while reducing the nanosystem complexity. In the light of the above considerations, hydrophobic alkaloid camptothecin (CPT, log P: 1.5, and MW: 364.5) and hydrophilic antimetabolite methotrexate (MTX, log P: -1.9 and MW: 454.5, easily soluble in phosphate buffer solution (PBS) at pH 7.4) were chosen as FDA approved anticancer drugs for synergistic combination chemotherapy.

23-24

CPT is an inhibitor of nuclear DNA

topoisomerase I and exhibits great antitumor activity against various types of malignant tumors through binding to DNA topoisomerase I and disrupting the 5 ACS Paragon Plus Environment


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structure and function of DNA. 25 Besides, MTX as an analogue of folic acid (FA) is an inhibitor of cytoplasmic enzyme (dihydrofolate reductase, DHFR) to treat various cancers through inhibiting folate metabolism and interrupting DNA and RNA synthesis. 26 Notably, as the most clinically used anticancer drugs, CPT and MTX possess different yet complementary anticancer mechanisms and distinct sites of action to reach the same goal.

27-28

Thus, if the combination therapy of

CPT and MTX was achieved, it could enhance the anticancer efficacy. Encouragingly, due to the high structural similarity between MTX and folate, regardless of a key feature that MTX had an amino group whereas FA had a hydroxyl group at the 4-position of pteridine ring (Scheme Scheme 1A), 1A MTX itself has been reported to be a potential specific tumor-targeting ligand for folate receptors uniquely overexpressed on most solid tumors and cancer cell lines. 29-30

Although folate receptors have lower affinities for MTX compared to that for

FA, the use of MTX as a ligand is still effective for tumor-targeting delivery if a multivalent design strategy is utilized which could offer very tight binding interaction compared to a weak monovalent binding.

31-35

Furthermore,

scientists have discovered that the free MTX molecules enter cancer cells via reduced folate carrier,

36

whereas Baker,

37

Wagner,

38

and Linden’s

29

groups

reported that the multivalent MTX surface-functionalized nanomaterials were specifically taken up by cancer cells via folate receptor-mediated endocytosis. In addition, to guide the therapeutic process, track the delivery of nanomedicines, and evaluate the efficacy of cancer treatment, there has been an increasing interest focusing on imaging modalities, such as near-infrared fluorescence (NIRF) imaging, magnetic resonance (MR) imaging, photoacoustic (PA) imaging, computed tomography (CT) imaging, ultrasound (US) imaging, etc. 39 However, a single-modal imaging still faces enormous challenges such as limited tissue penetration depth, low contrast, low sensitivity, and poor spatial resolution.

40

Thus, multimodal imaging is expected to compensate the intrinsic limitations of 6 ACS Paragon Plus Environment

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each single-modal imaging and considered as an inevitable trend in the development of new imaging techniques. 40-41 A good example is the integration of NIRF and PA, which could offer a real-time, comprehensive, and exact manner for cancer theranostics. 41-42 In this work, the two different anticancer drugs CPT and MTX were conjugated through ester bond to synthesize the MTX-CPT conjugate as one molecule. Due to its amphiphilic and ionic characteristics, the small molecular drug conjugate could be directly self-assembled into almost 100% drug loading nanostructures (MTX-CPT NPs) in aqueous solution, offering the advantages of favorable

pharmacokinetics

of

nanomedicine,

self-targeting

specificity,

controlled release, and synergistic drug combinations with a precise drug-to-drug ratio (Scheme Scheme 1). 1 On the benefit of the aforementioned advantages, the MTX-CPT NPs could be passively self-delivered to the tumor sites through the EPR effect.

14

More importantly, MTX would greatly enhance the cellular

internalization of MTX-CPT NPs via receptor-mediated endocytosis. Under the tumoral endo/lysosomal environment, the ester linkage in conjugate could be rapidly cleaved by acid hydrolysis and/or enzymolysis to release dual-drug synchronously at a fixed ratio. Furthermore, due to the different sites of pharmacological action and anticancer pathways of dual-drug, it is expected that the MTX-CPT NPs would exert effectively synergistic anticancer effect compared with both free drugs combination and individual free drug. 1’-dioctadecyl-3, 3, 3’, 3’-tetramethylindotricarbocyanine iodide (DiR) as a lipophilic and hydrophobic near-infrared (NIR) cyanine dye 43-44 was doped within MTX-CPT NPs to prepare the DiR-loaded MTX-CPT NPs for in vivo NIRF and PA dual-modal imaging. 2. RESULTS RESULTS AND DISCUSSION 2.1 Synthesis of MTXMTX-CPT Conjugate To prepare the small molecular NPs, amphiphilic MTX-CPT conjugate was designed and synthesized as shown in Figure 1A, B. B MTX-CPT was obtained via 7 ACS Paragon Plus Environment


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esterification of CPT and MTX using DCC/DMAP as a catalyst. The identities of MTX-CPT were confirmed by spectroscopy,

Fourier

1H

nuclear magnetic resonance (1H NMR)

transform

infrared

(FT-IR)

spectroscopy,

ultraviolet-visible (UV-vis) absorption spectroscopy, fluorescence spectroscopy, and high resolution mass spectroscopy (MS) (Figure Figure 1C1C-G). As shown in Figure 1C, 1C compared to the 1H NMR spectra of CPT, MTX, and MTX/CPT mixture, the proton signals at ∼10.48 ppm and ∼12.28 ppm respectively associated with hydroxyl of CPT and carboxyl of MTX disappeared in MTX-CPT conjugate because of the formation of ester linkage. Furthermore, the peak at ∼2.22 ppm related to -CH2- of MTX shifted to ∼1.88 ppm in MTX-CPT conjugate, which gave a proof that -CH2-CH2-COOH of MTX was reacted with the -OH group of CPT. Furthermore, compared to the FT-IR spectra of CPT and MTX/CPT mixture (Figure Figure 1D and Figure S1 in the Supporting Information), a high-wavenumber shift of -C=O stretching absorption band (carboxyl stretching vibration) from 1, 719 cm-1 to 1, 736 cm-1 was observed from MTX-CPT conjugate because of the newly generated ester bond. As shown in Figure 1E, 1E both characteristic absorptions of CPT and MTX could be observed from the UV-vis spectrum of MTX-CPT conjugate. Compared to the absorption peak of CPT and MTX/CPT mixture, a blue shift of the absorption peak from 382 to 380 nm was observed from the absorption of MTX-CPT conjugate. In addition, a 12 nm red-shift in the absorption peak of MTX-CPT conjugate at 312 nm was observed, by contrast with the absorption peak of MTX and MTX/CPT mixture at 300 nm. The maximum fluorescence emission peak of CPT was 430 nm, while the maximum fluorescence emission peak of MTX-CPT conjugate obviously red-shifted to 456 nm because of the formation of ester linkages (Figure Figure 1F). 1F The result of high resolution mass spectroscopy confirmed that the molecular weight of MTX-CPT (m/z, [M-H]+) was 801.2651 (Figure Figure 1G), 1G which was in agreement


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with the calculated value (m/z, [M-H]+, 801.2667). These experimental results further demonstrated the successful synthesis of MTX-CPT conjugate. 2.2 Preparation and Characterization of MTXMTX-CPT NPs The high solubility and ionicity of MTX in PBS buffer could compensate the extreme hydrophobicity of CPT, so the amphiphilic MTX-CPT conjugate could directly self-assemble into NPs in aqueous solution such as PBS buffer (Figure Figure 1B). 1B In addition, other weak intermolecular interactions among the conjugates such as hydrogen bond interaction and π-π stacking interaction could possibly make the hydrophobic interactions stronger, leading to enhanced confomational stability. The MTX-CPT NPs were prepared by a solvent exchange method, and the self-assembly behavior was corroborated by measuring the critical aggregation concentration (CAC) value as around 2.3 μM (Figure Figure S2 in the Supporting Information). To further confirm the formation of MTX-CPT NPs from MTX-CPT conjugate, the dynamic light scattering (DLS), scanning electron microscopy (SEM), transmission electron microscopy (TEM), confocal laser scanning microscopy (CLSM), and atomic force microscopy (AFM) were performed to determine and visualize the size and morphology of MTX-CPT NPs. The SEM, TEM, and AFM images showed that the MTX-CPT NPs were monodispersed spherical in distilled (DI) water or PBS (pH 7.4) in shape with a mean diameter of around 80-90 nm (Figure Figure 2A2A-D and Figure S3S3-4 in the Supporting Information), which was consistent with the data from DLS (Figure Figure 2E and Figure S5 in the Supporting Information). The CLSM images clearly showed that both small drug moieties were uniformly distributed throughout the MTX-CPT NPs (Figure Figure 2C). 2C A small proportion of MTX moieties was located on the NPs’ surface, which could be expected to exert targeting effect for selective cellular uptake. Besides, in the 1H NMR spectrum of MTX-CPT NPs, the proton signals (6.6, 7.7, and 8.8 ppm) of MTX molecules ascribing to the aromatic ring were detected in D2O solution, 9 ACS Paragon Plus Environment


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whereas most of the proton signals of CPT molecules disappeared (inset of Figure 2A), 2A which further confirmed the presence of hydrophilic MTX molecules on the surface of MTX-CPT NPs. The DLS analysis revealed a narrow monomodal distribution with a small mean hydrodynamic diameter of ∼110 nm (polydispersity index (PDI): 0.169), which was within the accepted range for efficiently enabling the EPR effect, and ensuring the passive tumor targeting in the body.

45-46

The zeta potential indicated that the MTX-CPT NPs displayed a

negative surface charge of ∼-25 mV (Figure Figure 2F). It is well-known that the CPT molecules tend to form needle shaped crystals due to the crystal growth along the edge direction. 27, 47 MTX-CPT is a conjugate with the 10-OH of CPT connected to a glutamate side chain of MTX through an ester linkage (Figure Figure 1). The insertion of dynamically rotated glutamate side chain into CPT could provide a more flexible steric “structures defects” to CPT, prevent long-range order of drug arrangement, and reduce the ability of crystallization of MTX-CPT conjugate. 48 The MTX-CPT conjugate and lyophilized powder of MTX-CPT NPs (DLS data was shown in Figure S5B) S5B were lack of apparent diffraction peaks compared with CPT, MTX, and MTX/CPT mixture powder as evidenced by X-ray diffraction (XRD) spectra (Figure Figure S6), indicating amorphous structure and low crystallinity of MTX-CPT conjugate and MTX-CPT NPs. The result suggested that the decreased crystallinity of MTX-CPT compared with that of individual free drug and both free drugs might be a possible reason for MTX-CPT prodrug self-assembling NPs. 2.3 In Vitro Colloidal Stability Colloid stability is one of the most important factors for biomedical application of nanoscale drug delivery system.

48

The DLS measurements were

performed at a series of different time intervals to study the stability of NPs. No significant change in the hydrodynamic diameter, PDI, and zeta potential of MTX-CPT NPs were observed in water for 120 h (Figure Figure 2G and Figure S8, S9 in 10 ACS Paragon Plus Environment

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the Supporting Information). The hydrodynamic diameter of MTX-CPT NPs also kept unchanged in PBS buffer and cell culture medium (RPMI-1640) within 48 h (Figure Figure 2G, H). H Furthermore, the hydrodynamic diameter of these NPs incubated in 10% fetal bovine serum (FBS) for 48 h showed little increase due to some level of protein adsorption (Figure Figure 2G, 2G, H, and Figure S1010-11 in the Supporting Information). Thus, although no surfactants or excipients were used, the MTX-CPT NPs showed excellent stability without exhibiting any precipitation and phase separation in water, PBS, cell culture medium, and serum. In addition, the high stability of nanoassemblies of entirely hydrophobic drug (paclitaxel) dimers in PBS or PBS containing FBS in the absence of surfactants (such as PEG) has been reported elsewhere.

48

The result indicated that the MTX-CPT NPs

might arrive to tumor sites in intact form during circulation, which would have a great potential in passive-plus-active tumor-targeting delivery. 2.4 2.4 In Vitro Drug Release The in vitro release behaviors of dual drugs from MTX-CPT NPs were tested by a dialysis method in simulated physiological (pH 7.4) and acidic (pH 5.0) condition in absence and presence of esterase (30 U/mL, Aldrich chemicals) at 37°C. As shown in Figure 2I, J, the MTX-CPT NPs exhibited obvious pH-/enzyme-responsive release behaviors of dual drugs. At pH 7.4 without esterase, only ~20% of CPT and ~25% of MTX were released over 24 h, indicating that the MTX-CPT NPs still remained a relatively high stability in the physiological condition. However, the cumulative release of CPT and MTX from MTX-CPT NPs was more than ~40% and ~50%, respectively at pH 5.0 over 24 h, indicating the free form of CPT and MTX were released more rapidly from NPs in the acidic condition. Furthermore, it is worthwhile pointing out that the introduction of esterase remarkably promoted the release rate of both CPT and MTX in both pH conditions. In particular, at pH 5.0 with esterase, the cumulative release of CPT and MTX was up to ~60% and ~70% over a period of 24 h, 11 ACS Paragon Plus Environment


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respectively. These results were consistent with the reported literatures that ester linkage could be cleaved much more quickly by both acid and esterase conditions.

14

In addition, the faster release rate of MTX than CPT was

presumably due to their differences in aqueous solubility because the solubility of MTX in PBS was higher than CPT. Therefore, it was expected that once the MTX-CPT NPs were internalized by cancer cells inside acidic endo/lysosomes, abundant of esterases with the help of acid in the endo/lysosomes could easily accelerate the hydrolysis of ester bond connected MTX and CPT and release two pharmaceutically/pharmacologically active agents. The pH-/esterase-responsive release behavior might specifically cause the disassembly of nanostructure (DLS results and TEM images were seen in Figure 2K, L and Figure S12 in the Supporting Information, the partial disassembly and aggregation of NPs was attributed to the cleavage of ester linker and the aggregation of hydrophobic CPT

49)

and enhance the drug release in

acidic endo/lysosomal environment of tumor cells, while reducing the undesired side effects to normal cells during circulation in the body. In addition, the MTX-CPT NPs exhibited very weak fluorescence in water and PBS (pH 7.4) (Figure Figure 2M 2M, N), since the inherent fluorescence of CPT was suppressed by the self-assembly into nanostructure. By contrast, the strong fluorescence was observed when the MTX-CPT NPs was introduced into PBS (pH 5.0) or DMSO solution. The fluorescence recovery might be due to the fact that the cleavage of ester bond in MTX-CPT conjugate in the acidic condition induced the disassembly of MTX-CPT NPs. This interesting phenomenon indicated that the self-assembled MTX-CPT NPs exhibited a fluorescence “turn-off” effect, whereas the disassembly of MTX-CPT NPs in acidic environment or organic solvent (e.g., DMSO) resulted in a fluorescence “turn-on” effect. Thus, the MTX-CPT NPs could also serve as a fluorescence switch for real-time monitoring drug release by cell


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imaging (Figure Figure 2O). 2.5 2.5 In Vitro Cellular Uptake To prove our assumption that the MTX-CPT NPs could provide specificity to target cancer cells, HeLa human cervical carcinoma cells (high expression of folate receptors) and A549 human lung carcinoma cells (low expression of folate receptors) were incubated with MTX-CPT NPs for 1 and 4 h, both CLSM (the cells were washed and fixed by 70% ice-cold ethanol, then the cells were washed and the nucleus were then counterstained by propidium iodide (PI, blue-false color) 50)

and flow cytometry analysis were performed by determining the inherent

fluorescence of CPT. As shown in Figure 3A3A-C, the fluorescence intensity of HeLa cells was significantly stronger compared to A549 cells since the specific binding affinity of MTX-CPT NPs surface’s MTX ligands (see inset of Figure 2A and 3C) 3C towards folate receptors overexpressed on the surface of HeLa cells membrane could result in an enhanced uptake of MTX-CPT NPs. Furthermore, the petreatment of HeLa cells with free FA molecules significantly inhibited the cellular uptake efficiency of MTX-CPT NPs instead of MTX/CPT mixture (Figure Figure 4A, B). B It was due to the competitive binding affinity of folate receptors on the cell membrane to free FA molecules which blocked the folate receptor-mediated endocytosis of MTX-CPT NPs. 31, 33-34 The above results of competitive inhibition assay proved that the active cellular uptake was mainly induced by the highly specific multivalent interaction

51-52

at the interface of NPs’ multivalent MTX

ligands and cell membranes surface’s multivalent folate receptors. With respect to the MTX/CPT mixture, both CPT and MTX drug molecules entered into cells by passive diffusion and via reduced folate carrier (active transport system)

36,

respectively. Whereas, MTX-CPT NPs were internalized into the endo/lysosomes (inset inset of Figure 4C) 4C by folate receptor-mediated active endocytosis. This also explained that the cellular uptake efficiency of MTX-CPT NPs was higher than that of MTX/CPT mixture (Figure Figure 4C). 4C 13 ACS Paragon Plus Environment


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In addition, the treatment with low temperature (4°C) drastically decreased the intracellular uptake of MTX-CPT NPs (Figure Figure S13 in the Supporting Information), indicating an energy-dependent endocytosis of MTX-CPT NPs. Thus, with the help of MTX-targeting carrier-free nano-multi-drug, drugs could be

specifically

and

energy-dependently

self-delivered

to

folate

receptor-overexpressing cancer cells, which is beneficial for alleviating side effects. 2.6 2.6 Endocytosis and Subcellular Localization The intracellular drug distribution, delivery, and release of MTX-CPT NPs were further investigated using CLSM (Figure Figure 5). To image the intracellular distribution of MTX in living cells, NBD·Cl with a very small molecular structure was used for fluorescence labeling. HeLa cells were treated with NBD labeled MTX-CPT NPs (NBD-MTX-CPT NPs) at the equivalent concentration of dual drugs for different incubation time periods (1, 4, 8, and 16 h). It is worth noting that the fluorescence signals of CPT became significantly stronger and moved from acidic endo/lysosomes via the cytoplasm into nucleus (the site of action of CPT). Besides, the fluorescence signals of NBD-MTX became stronger and distributed in the cytoplasm (the site of action of MTX). The above-mentioned results suggested that the targeted cellular uptake and co-localization with acidic endo/lysosomes could trigger the burst release of both drugs at a precise ratio from these pH-responsive

MTX-CPT NPs, resulting in rapid nuclear

internalization of CPT, efficient cytoplasmic accumulation of MTX, and thus effectively synergistic tumor cell killing (Figure Figure 6A). 6A Meanwhile, the MTX-CPT NPs performed great cell imaging ability, which could real-time monitor the intracellular drug delivery and drug release of MTX-CPT NPs. 2.7 2.7 In Vitro Cytotoxicity To evaluate the inhibition effect of cellular proliferation of MTX-CPT NPs against folate receptor-overexpressing cancer cells, the cell viability was 14 ACS Paragon Plus Environment

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assessed by a Cell Counting Kit-8 (CCK-8) assay. HeLa cells and MCF-7 human breast carcinoma cells (high expression of folate receptors) were treated with free MTX, free CPT, MTX/CPT mixture, or MTX-CPT NPs at a series of concentrations (0.25, 0.5, 1, 2, 4, 8, 16, 32, and 64 μM) for 48 h incubation. As shown in Figure 6B, 6B, C the MTX-CPT NPs exhibited the remarkably higher cellular proliferation inhibition towards cancer cells than free MTX, free CPT, and MTX/CPT mixture. Also, IC50 values of all formulation in HeLa and MCF-7 cells were calculated in Table S1, 2 in the Supporting Information. These results indicated the MTX-CPT NPs exhibited the significantly more effective anticancer activity compared to other three drug formulations. Although the MTX/CPT mixture contained the same drug dosage as the MTX-CPT NPs, it exhibited less cytotoxicity due to the poor cellular uptake and the unfixed drug-to-drug ratio uptaken by cells. In addition, the pre-treatment of HeLa cells with free FA resulted in a significant decrease of anticancer activity for MTX-CPT NPs (Figure Figure 6D), 6D confirming folate receptor-mediated endocytosis of MTX-CPT NPs by HeLa cells. Morerover, the MTX-CPT NPs showed slightly lower anticancer activity in A549 cells than MTX/CPT mixture (Figure Figure S14 in the Supporting Information), which might be attributed to the low expression of folate receptors on A549 cells. It was clear that the self-assembled MTX-CPT NPs could be specifically selectively and uptaken by folate receptor-overexpressing cancer cells, as well as efficiently release and deliver a fixed ratio of both drugs to exert the synergistic action. 2.8 2.8 In Vitro Synergistic Effect To further confirm the synergy in the MTX-CPT NPs, the combination index (CI) method was carried out. 53 As shown in Figure 6E, 6E the CI values of MTX-CPT NPs were much lower than those of MTX/CPT mixture at each corresponding ICx value, demonstrating that the synergistic effect of MTX-CPT NPs was more remarkable than just the free small molecular drug combination at the same 15 ACS Paragon Plus Environment


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drug dose. The data strengthened the above explanation that, after selective internalization into cancer cells by folate receptor-mediated endocytosis, CPT and MTX released from self-targeting MTX-CPT NPs inside enzyme-rich acidic endo/lysosome could still remain the same molar ratio of 1:1. Thus, both CPT and MTX self-co-delivered by MTX-CPT NPs with the fixed drug-to-drug ratio could interact with their individual sites of action (see inset of Figure 6E, 6E the sites of pharmacological action of CPT and MTX were cytoplasm and nucleus, respectively) and synergistically resulted in highly efficient anticancer activity. 2.9 In Vitro Apoptosis In order to demonstrate the apoptosis-inducing capability of MTX-CPT NPs, the Annexin V-FITC/PI apoptosis detection was performed to compare that of individual free drug group and both free drugs group. After treatment with the free MTX, free CPT, free MTX/CPT, and MTX-CPT NPs for 24 h incubation, the total apoptotic ratio obtained by a sum of the early apoptotic ratio and the late apoptotic ratio of HeLa cells was ∼15.4%, ∼24.0%, ∼30.2%, and ∼56.9%, respectively (Figure Figure 6F and Figure S15 in the Supporting Information). The result exhibited that the MTX-CPT NPs induced significantly higher apoptotic ratio than individual free drug group and both free drugs group at the equivalent drug concentration. Thus, consistent with the in vitro cytotoxicity and synergistic effect, the results demonstrated that the enhanced cellular uptake by folate receptors-mediated endocytosis and the simultaneous accelerated release of both drugs triggered by acidic pH/esterase resulted in remarkable enhancement of apoptosis-inducing activity. 2.10 2.10 In Vivo NIRF/PA Dualual-Modal Imaging The

in

vitro

results

regarding

the

self-recognition

to

folate

receptor-overexpressing cancer cell lines stimulated us to evaluate the in vivo tumor-targeting ability of MTX-CPT NPs toward folate receptor-overexpressing solid tumors. By doping with a lipophilic NIR cyanine dye (DiR with strong NIR 16 ACS Paragon Plus Environment

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absorbance and fluorescence), the DiR-loaded MTX-CPT NPs could be an Figure 7A). effective probe for in vivo NIRF/PA dual-modal imaging (Figure 7A HeLa tumor-bearing BALB/c nude mice were used as the animal modal and intravenously injected with free DiR and DiR-doped MTX-CPT NPs, respectively. Subsequently, the in vivo fluorescence images were acquired at predetermined time intervals to monitor the in vivo biodistribution of MTX-CPT NPs. Interestingly, in the case of DiR-doped MTX-CPT NPs-treated mice, the efficient tumor accumulation of fluorescence signals was observed at just 4 h post-injection and the fluorescence signals in tumor tissue continued to increase and reached a peak after 24 h post-injection (Figure Figure 7B). 7B On the contrary, the fluorescence signals mainly distributed within liver after intravenous injection of free DiR into mice. Furthermore, the much stronger fluorescence at the tumor site was still observed from DiR-doped MTX-CPT NPs group in comparison with free DiR group even at 24 h post-injection. The result indicated that the MTX-CPT NPs could reduce the rapid capture by the reticuloendothelial system (RES) and passively accumulate in the tumor by the EPR effect. In addition, the PA signals visually revealed the accordant distribution of DiR within and outside the tumor microvessels.

42

The PA imaging based on DiR-doped MTX-CPT NPs provided a

high spatial resolution, which showed the perfect accumulation of DiR in tumor tissue and provided information on tumor microstructure (Figure Figure 7B and Figure S16 in the Supporting Information). After 24 h post-injection, the tumor and main organs of mice were further isolated for ex vivo NIRF imaging. As show in Figure 7C, 7C, D, D most of DiR accumulated in the liver after intravenous injection of free DiR into mice. Furthermore, the tumor in DiR-doped MTX-CPT NPs groups showed significantly stronger DiR fluorescence compared to that in free DiR group. It should be noted that some level of fluorescence was also observed in other important metabolic organs including the liver, which has been reported for most conventional drug 17 ACS Paragon Plus Environment


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delivery systems with nanoscale characteristic due to the RES uptake and was a universal phenomenon for nanomedicines themselves. CLSM images obtained from frozen tumor tissue further confirmed highly effective tumor accumulation of MTX-CPT NPs (Figure Figure 7E). These data indicated the MTX-CPT NPs could preferentially accumulate at the tumor site by the EPR effect-mediated passive targeting and folate receptor-MTX ligand interaction-mediated active targeting. 54-56

In addition, the biodistribution of MTX-CPT NPs in the normal organs and tumors was detected by HPLC measurements. As shown in Figure S17 S17 in the Supporting Information, at 24 h post-injection, the drug level in tumor reaches ~6.5% ID/g, which was about 13.0 and 9.3 times higher than CPT (~0.5% ID/g) and MTX (~0.7% ID/g), respectively. The higher tumor-to-normal tissue distribution ratio of MTX-CPT NPs compared to CPT and MTX indicated the remarkably enhanced tumor selectivity of MTX-CPT NPs. The in vitro cellular uptake behaviors of MTX-CPT NPs and in vivo imaging results concluded that the MTX-CPT NPs could efficiently co-deliver both CPT and MTX to the tumor site, which

was

likely

attributed

to

both

the

EPR

effect

and

folate

receptor-co-mediated passive-plus-active tumor targeting mechanisms. 54-58 2.1 2.11 In Vivo Blood Circulation It is generally considered that NPs with a suitable size (smaller than 200 nm) exhibit a longer retention time in the bloodstream compared with free small molecular drugs.

1,

5,

59-61

To confirm this hypothesis, The in vivo

pharmacokinetic studies were performed by determining the plasma drug concentration at predetermined time points after intravenous injection of MTX, CPT, and MTX-CPT NPs (4 mg/kg) into Sprague-Dawley (SD) rats (~200 g) (Figure Figure 7F). 7F Notably, it could be seen that the blood circulation half-life of MTX-CPT NPs without the help of any drug carriers was significantly prolonged comparable to free MTX or CPT, indicating a longer blood retention time of 18 ACS Paragon Plus Environment

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MTX-CPT NPs. Thus, the significantly improved retention in bloodstream and more favorable pharmaceutics would provide the possibility of enhanced accumulation of MTX-CPT NPs at the tumor site (see Figure 7B7B-E). 2.12 2.12 In Vivo Antitumor Effect Studies To evaluate whether effective tumor accumulation and improved biodistribution of MTX-CPT NPs resulted in the improvement of therapeutic performance, HeLa tumor-bearing nude mice were intravenously administrated with 0.9% NaCl as control, MTX, CPT, MTX/CPT mixture, and MTX-CPT NPs via the tail vein on days 0, 3, 6, and 9. Tumor size and body weight of HeLa tumor-bearing nude mice were monitored every 3 days for 21 days. On day 21, the relative tumor volumes in MTX-CPT NPs-treated mice were remarkably smaller compared with those in mice treated with 0.9% NaCl, MTX, CPT, and MTX/CPT mixture, respectively (Figure Figure 8A), 8A which demonstrated that the MTX-CPT NPs showed the strongest inhibition effect of tumor growth in all therapeutic groups. At the end of the experiments, the HeLa tumor-bearing nude mice were sacrificed and tumors were dissected. Figure 8B, C confirmed that the HeLa tumor-bearing nude mice treated with MTX-CPT NPs had the smallest tumor size and tumor weight. In addition, the inhibition rate of tumor growth of MTX-CPT NPs group was significantly higher than that of either individual free drug group or both free drugs group (Figure Figure S18 S18 in the Supporting Information). These results further demonstrated that the MTX-CPT NPs group significantly therapeutic efficacy compared to the other therapeutic groups. Meanwhile, more than 15% loss of body weight could be observed in the free CPT or MTX/CPT mixture group (Figure Figure 8D). 8D By contrast, the treatment with MTX-CPT NPs had no significant influence on the body weight of mice, indicating that MTX-CPT NPs had low systemic side effects. 2.13 Histological Analyses 19 ACS Paragon Plus Environment


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Histological analyses of hematoxylin and eosin (H&E) staining of tumor tissue section was used to evaluate the antitumor effect after the treatment of the HeLa tumor-bearing nude mice with different formulations. The much more irregularly shaped necrotic area with cellular shrinkage and nuclear fragmentation was obviously observed from the MTX-CPT group (Figure Figure 8E). 8E In comparison, the tumor cells in the tumor tissues partially or largely retained their regular cell morphology with intact cell nucleus in the other therapeutic groups. Furthermore, the H&E staining demonstrated that the MTX-CPT NPs caused low side effects to normal organ tissues (Figure Figure 8F). 8F The low liver damage was surprising as some level of MTX-CPT NPs were shown to accumulate in the liver. The H&E staining results further confirmed the superior

in vivo antitumor efficacy of MTX-CPT NPs with the reduced systemic toxicity. Therefore, the robust MTX-CPT NPs could mediate efficient and tumor-selective targeted delivery of dual drugs, leading to the efficiently improved therapeutic outcome while reducing the side effect. 2.14 In Vivo Safety Studies In order to evaluate the hemocompatibility of MTX-CPT NPs, the hemolysis assay was performed. As shown in Figure S19 S19 in the Supporting Information, the MTX-CPT NPs exhibited no significant red cell membranes damage even at the concentration of 1 mM (equivalent to a dose of 8 mg/kg), while the free MTX or CPT showed obvious hemolysis compared with the positive control (Triton X-100). In addition, in order to examine the hepatic and renal toxicity, the hematological and blood biochemical analyses were performed. The healthy BALB/c mice were intravenously injected with 0.9% NaCl or MTX-CPT NPs on days 0, 3, 6, and 9. As shown in Figure S20, 20, S2 S21 in the Supporting Information, at 16 days post-initial injection, all measured factors including alanine transaminase (ALT), aspartate transaminase (AST), blood urea nitrogen (BUN), and creatinine (CREA) levels of MTX-CPT NPs groups showed no statistically 20 ACS Paragon Plus Environment

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significant (P > 0.05) difference with those in 0.9% NaCl group. The results indicated that the MTX-CPT NPs induced no obvious inflammation reaction and noticeable hepatic and renal dysfunction. 3. CONCLUSION On the basis of the self-assembly of dual-acting MTX-CPT conjugate, a novel kind of tumor-selective self-targeting nano-multi-drug delivery system was developed. Unlike the simultaneous administration of two free chemotherapeutic drugs, the MTX-CPT NPs with pH-/esterase-responsive release property could keep synergistic drug-to-drug ratio for prolonged time and specifically co-deliver both CPT and MTX into folate receptor-overexpressing cancer cells. Furthermore, based on NIRF/PA dual-modal imaging, the DiR-doped MTX-CPT NPs showed the highly efficient tumor accumulation via both EPR effect-mediated passive targeting and folate receptor-mediated active targeting. More importantly, the systematical in vitro and in vivo studies suggested that the MTX-CPT NPs exhibited the superior synergistic effect, and could improve the therapeutic efficiency significantly with reduced toxicity compared to either individual free drug or combination of both free drugs. The rationale of the combinational effect from “an early-phase tumor-selective self-targeting and a late-phase synergistic anticancer” could give inspiration to design new “all-in-one” nanodrugs for collaboration and cooperation of self-targeted drug delivery and synergistic combination chemotherapy. ASSOCAITED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Experimental section and additional tables (Table S1-2)/figures (Figure S1-S21), including IC50 values of different drug formulations, FTIR/XRD spectra, SEM/AFM images, DLS/zeta potential/PDI data, stability results, CLSM images, schematic route of synthesis of NBD-MTX, cell viability assay, apoptosis assay, PA 21 ACS Paragon Plus Environment


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intensity

analysis,

in

vivo

biodistribution,

hemolysis

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assay,

blood

biochemical/haematological analysis, and so forth (PDF). AUTHOR INFORMATION Corresponding Author * Zhenqing Hou. E-mail address: [email protected] * Fanghong Luo. E-mail address: [email protected]. Author Contributions ‡ Dr.

Y. Li and J. Lin contributed equally to this work.

Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS The work is supported by the National Natural Science Foundation of China (No. 31771029), the Special Funding of China Postdoctoral Science Foundation (2017T100472), the China Postdoctoral Science Foundation (2016M602074), the Natural Science Foundation of Fujian Province of China (2016J01406), the Clinical Medicine Science and Technology Project of Jiangsu Province (BL2013015), and the Science and Technology Plan Guidance Project of Xiamen City (3502Z20159001).

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Figures Figures and captions in Manuscript

Scheme 1. (A) Optimized molecular structures and chemical structures of FA and MTX. MTX is an analogue of FA because of structural similarity, regardless a key feature that MTX has an amino group whereas FA has a hydroxyl group at the 4-position of pteridine ring. (B, C) Schematic illustration of MTXMTX-CPT nanodrug for the selfself-assembly assembly, bly, selfself-active targeted multimulti-drug coco-delivery, and synergistic anticancer effect: (I, II) MTX-CPT NPs accumulated at the tumor site by passive (EPR effect)-plus-active (folate receptor-mediated endocytosis) targeting mechanisms. (III) Release of both MTX and CPT was accelerated by acidic pH and esterase in intracellular endo/lysosomes. (IV) Free CPT was diffused into the nucleus to bind to DNA strands and free MTX was diffused into the cytoplasm to bind with DHFR enzyme, thus synergistic anticancer mechanism was realized by induction of cellular apoptosis/death of both CPT and MTX via disrupting DNA structure and function and interrupting DNA synthesis, respectively. 28 ACS Paragon Plus Environment

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Figure 1. Synthesis and characterization of MTXMTX-CPT conjugate. (A) Synthesis route of MTX-CPT conjugate through esterification. (B) Schematic illustration of synthesis of MTX-CPT conjugate, self-assembly of MTX-CPT conjugate, and pH-/esterase-responsive drug release and sequential function “Targeting-Anticancer” switch/fluorescence “Off-On” switch of self-targeting MTX-CPT NPs. Multivalent MTX moieties on the MTX-CPT NPs’ surface, not only could act as self-targeting ligands that multivalently bind with the folate receptors to enhance the cellular uptake, but also could serve as anticancer drugs that tightly bind with the DHFR enzyme to induce the cellular apoptosis/death. (C) 1H NMR spectra (DMSO-d6 as solvent), (D) FT-IR spectra, (E) UV-vis-NIR absorbance spectra (DMSO as solvent), and (F) fluorescence emission spectra (DMSO as solvent) of MTX, CPT, MTX/CPT mixture, and MTX-CPT conjugate. (G) MS spectrum of MTX-CPT conjugate. The molecular weight of MTX-CPT (m/z, [M-H]+) is 801.2651, which is well in line with the calculated value (m/z, [M-H]+, 801.2667). 29 ACS Paragon Plus Environment


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Figure 2. Characterization and and dualdual-drug release of MTXMTX-CPT NPs. (A) SEM and (B) TEM images of MTX-CPT NPs dispersed in DI water and PBS (pH 7.4). Inset: 1H

NMR spectra of MTX-CPT conjugate in DMSO-d6 and MTX-CPT NPs in D2O. (C)

TEM and CLSM images of NBD-MTX-CPT NPs dispersed in DI water and PBS (NBD-MTX was coupled via a simple substitution reaction between NBD·Cl and aromatic amino group of MTX (see Figure S7 in the Supporting Information), micro-sized particles could be formulated under low amplitude and high drug concentration). CPT: false-color green, NBD-MTX: false-color red. (D) Photographs of free CPT (2 mg/mL) precipitated in PBS (0.2 M, pH 7.4), free MTX (2 mg/mL) disolved in PBS (0.2 M, pH 7.4), and MTX-CPT NPs (2 mg/mL) dispersed in PBS (0.2 M, pH 7.4). (E) Hydrodynamic diameter (Dh) of MTX-CPT NPs dispersed in PBS (pH 7.4). (F) Zeta potential of MTX-CPT NPs dispersed in DI water. (G) Change of hydrodynamic diameter (Dh) and zeta potential of MTX-CPT NPs in water, PBS, and cell culture medium, and serum for 120 h. (H) Hydrodynamic diameter distributions of MTX-CPT NPs in water, PBS, and cell culture medium, and serum after incubation for 48 h. (I, J) In vitro release kinetics of (I) CPT and (J) MTX from MTX-CPT NPs in buffer (pH 7.4 and 5.0 with/without the presence of esterase) at 37°C. (K) pH-responsive disassembly and (L) enzyme-responsive disassembly of of MTX-CPT NPs determined by change of hydrodynamic diameter (Dh). (M) Fluorescence emission spectra and (N) normalized fluorescence intensity of MTX-CPT NPs in different media. Inset: photographs under UV light at 365 nm. (O) Detailed schematic illustration of pH-responsive drug release and sequential fluorescence “Off-On” switch of MTX-CPT NPs. Error bars indicate SD (n = 3).



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Figure 3. In vitro cell uptake of MTXMTX-CPT NPs. (A) CLSM images of HeLa cells (high expression of folate receptors) and A549 cells (low expression of folate receptors) treated with MTX-CPT NPs for 1 and 4 h. After incubation, the cells were washed and fixed by 70% ice-cold ethanol. Then the cells were washed with PBS and the nucleus were counterstained by propidium iodide (PI, blue-false color). CPT: false-color green. For CPT: λex = 405 nm, band-pass filter λ = 500-550 nm. For PI: λex = 543 nm, band-pass filter λ = 575-625 nm. Error bars indicate SD (n = 4). (B, C) Flow cytometry profiles and mean fluorescence intensity of HeLa cells treated with MTX-CPT NPs for 1 and 4 h.


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Figure 4. Mechanisms of competitive inhibition of MTXMTX-CPT NPs. (A) Confocal laser scanning microscopic (CLSM) images and (B) flow cytometry profiles of HeLa cells incubated with MTX/CPT mixture and MTX-CPT NPs with/without the pre-treatment of free FA for 2 h. After incubation, the cells were washed and then fixed by 70% ice-cold ethanol. Then the cells were washed with PBS and the nucleus were counterstained by PI (blue-false color). CPT: false-color green. For CPT: λex = 405 nm, band-pass filter λ = 500-550 nm. For PI: λex = 543 nm, band-pass filter λ = 575-625 nm. (C) Mean fluorescence intensity of MTX/CPT mixture or MTX-CPT NPs internalized by HeLa cells after incubation for 0.5, 1, 1.5, and 2 h. Error bars indicate SD (n = 4). *P < 0.05. Inset: Subcellular location of MTX-CPT NPs in HeLa cells after incubation for 2 h. Lysotracker Red (false-color red) was used to identify endo/lysosomes. For CPT: λex = 405 nm, band-pass filter λ = 430-480 nm. For Lysotracker Red: λex = 543 nm, band-pass filter λ=580-650 nm.The majority of CPT fluorescence was found to be co-localized in the acidic endo/lysosome after 2 h incubation of MTX-CPT NPs, indicating that almost all the MTX-CPT NPs were internalized into the endo/lysosomes via the endocytosis pathway.



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Figure 5. SpatialSpatial-temporal dualdual-drug distribution of HeLa cells treated with NBD--labeled MTXNBD MTX-CPT NPs (NBD(NBD-MTXMTX-CPT NPs). The images were taken immediately (fluorescence “Off”), 1 h (fluorescence “On”), 4 h, 8 h, and 16 h after adding MTX-CPT NPs to HeLa cells. NBD-MTX was coupled via a simple substitution reaction between NBD·Cl and aromatic amino group of MTX. After incubation, the cells were washed and then fixed by 70% ice-cold ethanol. Then the cells were washed with PBS and the nucleus were then counterstained by PI (blue-false color). CPT: false-color green. NBD-MTX: false-color red. For PI: λex = 543 nm, band-pass filter λ = 575-625 nm. For CPT: λex = 405 nm, band-pass filter λ = 500-550 nm. For NBD-MTX: λex = 488 nm, band-pass filter λ=500-540 nm. 34 ACS Paragon Plus Environment

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Figure 6. Intracellular dualdual-drug delivery and cytotoxicity of MTXMTX-CPT NPs. (A) CLSM images of HeLa cells treated with CPT, NBD-MTX, or NBD-MTX-CPT NPs for 24 h incubation. (B, C) Cell viability of (B) HeLa cells and (C) MCF-7 cells treated with MTX, CPT, MTX/CPT mixture, or MTX-CPT NPs for 48 h incubation. (D) Cell viability of HeLa cells treated with MTX-CPT NPs with/without pre-treatment of free FA for 48 h incubation. (E) Combination index (CI) of MTX and CPT combinations via MTX/CPT mixture or MTX-CPT NPs against HeLa cells for 48 h incubation. CI values 1 indicate antagonism. Inset: intracellular distribution of CPT and MTX delivered by MTX-CPT NPs in nucleus and cytoplasm, respectively after 48 h incubation. Error bars indicate SD (n = 4). (F) Apoptosis analysis of HeLa cells detected by flow cytometry induced by MTX, CPT, MTX/CPT mixture, or MTX-CPT NPs for 24 h incubation using Annexin V-FITC/PI Apoptosis Detection Kit. The lower-left (Annexin V-FITC-, PI-), upper-left (Annexin V-FITC-, PI+), lower-right (Annexin V-FITC+, PI-), and upper-right (Annexin V-FITC+, PI+) quadrants in flow cytometry graph represent normal cells, necrotic cells, early apoptotic cells, and late apoptotic cells, respectively. 35 ACS Paragon Plus Environment


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Figure 7. In vivo bimodal imaging of DiRDiR-doped MTXMTX-CPT NPs. (A) Schematic illustration of preparation of DiR-doped MTX-CPT NPs for NIRF/PA dual-modal imaging. (B) In vivo NIRF and PA images of the HeLa tumor-bearing nude mice after intravenous injection of free DiR or DiR-doped MTX-CPT NPs. (C) Ex vivo NIRF images and (D) semiquantitative biodistribution of the major organs and tumors excised at 24 h post-intravenous injection of free DiR or DiR-doped MTX-CPT NPs. (E) Frozen sections of tumors excised at 24 h post-intravenous injection of free DiR or DiR-doped MTX-CPT NPs. Nucleus was stained with PI. PI: false-color blue, DiR: false-color red. PI was excited by a 543 nm laser, while DiR was excited by a 633 nm laser. (F) In vivo pharmacokinetics profiles of free MTX, free CPT, and MTX-CPT NPs at the equivalent dose (4 mg/kg) in Sprague-Dawley (SD) rats. Error bars indicate SD (n = 3). **P < 0.01. 36 ACS Paragon Plus Environment

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Figure 8. In vivo therapeutic efficacy of HeLa tumortumor-bearing nude mice after intravenous injection of 0.9% NaCl, free MTX (4.54 mg/kg), free CPT (3.64 mg/kg), MTX/CPT mixture (8.18 mg/kg), or MTXMTX-CPT NPs (8.00 mg/kg) at the equivalent dose of MTX or CPT. (A) Tumor volumes changes, (B) tumor weight, (C) excised tumor volume, and (D) body weight changes. (E) Representative images of HeLa tumor of nude mice and H&E staining histological images obtained from tumor of HeLa tumor-bearing nude mice after different treatments. (F) Representative H&E staining histological images obtained from liver, spleen, lung, kidney, and heart of HeLa tumor-bearing nude mice after intravenous injection of 0.9% NaCl or MTX-CPT NPs. Error bars indicate SD (n = 5). *P < 0.05. **P < 0.01. 37 ACS Paragon Plus Environment


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