The Effect of Molecular Structure on Cytotoxicity and Antitumor Activity

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The Effect of Molecular Structure on Cytotoxicity and Antitumor Activity of PEGylated Nanomedicines Wenhai Lin, Lei Yin, Tingting Sun, Tingting wang, Zhigang Xie, Jingkai Gu, and Xiabin Jing Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b00083 • Publication Date (Web): 02 Apr 2018 Downloaded from http://pubs.acs.org on April 2, 2018

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The Effect of Molecular Structure on Cytotoxicity and Antitumor Activity of PEGylated Nanomedicines Wenhai Lin, †, ‡,# Lei Yin, §,∥,# Tingting Sun, †, ‡ Tingting Wang, §,∥ Zhigang Xie,*,† Jingkai Gu*,§,∥ and Xiabin Jing† †

State Key Laboratory of Polymer Physics and Chemistry, Changchun Institute of Applied

Chemistry, Chinese Academy of Sciences, 5625 Renmin Street, Changchun, Jilin 130022, P. R. China ‡

University of Chinese Academy of Sciences, Beijing 100049, P. R. China

§

Research Center for Drug Metabolism, College of Life Sciences, Jilin University, Changchun

130012, P. R. China ∥

Clinical Pharmacology Center, Research Institute of Translational Medicine, The First Hospital

of Jilin University, Dongminzhu Street, Changchun 130061, PR China #

These authors contributed equally.

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ABSTRACT: Fundamental studies on the cellular uptake and drug release of PEGylated nanomedicines are beneficial to understand their fate in vivo and construct ideal nanoparticle formulations. In this work, the detailed metabolic process of PEGylated doxorubicin (Dox) nanomedicines were investigated via confocal laser scanning microscopy (CLSM), flow cytometry (FCM), cytotoxicity test, fluorescence imaging in vivo (FLIV) and liquid chromatography tandem mass spectrometry (LC-MS/MS). Among them, only LC-MS/MS could determine accurately the content of PEGylated Dox and Dox in vitro and in vivo. To the best of our knowledge, it was the first time to quantify simultaneously the PEGylated Dox and released Dox. The interplay of molecular structures, cellular uptake, drug release and antitumor effect was well characterized. PEG with high molecular weight impeded the cellular uptake of nanoparticles, and the acid-labile hydrazone bond between Dox and PEG promoted Dox release significantly. Cellular uptake and drug release play decisive roles in cytotoxicity and antitumor effect, as evidenced by LC-MS/MS. We emphasized that LC-MS/MS would be a practicable method to quantify PEGylated drugs without complex tags, which could be more in-depth to understand the interaction between PEGylated nanomedicines and their antitumor efficacy.

KEYWORDS: poly(ethylene glycol), doxorubicin, nanomedicines, LC-MS/MS


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INTRODUCTION Nanoparticle formulations have been developed to increase the efficacy and reduce the systemic toxicity of therapeutic agents.1-5 A myriad of nanoparticles are reported and exhibited unique physicochemical properties and multifunctional characteristics for tumor treatment.6-11 Among these formulations, polymeric nanoparticles possess some unique features, including tunable biodegradability, variable molecular structures and multiple approaches for incorporating drug.1219

Although great progress is made in past decades, the successful clinical translation of

nanomedicines is limited.20 One possible reason is that some fundamental questions were not understood well yet, for example, the interactions of nanoparticles with cells and organs, and how to accurately determine nanoparticle delivery efficiency and drug release in living cells and animals.21,22 Poly(ethylene glycol) (PEG) conjugation, also known as PEGylation, is a versatile strategy to modify the drugs, which could form PEGylated nanomedicines to improve the solubility, increase circulation time and enhance therapeutic index.23-28 PEGylation had an important effect on protein absorption, blood circulation and tumor targeting.29-34 Understanding the interactions of nanoparticles and cells will provide important guidance for developing optimal nanosystems.35,36 Up to now, there are some ways for indirect quantification of cellular uptake of PEGylated nanoparticles, like hybridization of metal nanoparticles and tags of fluorescent dyes.37-39 Parak et al. reported that the high molecular weight PEG coating led to uptake reduction of PEGylated nanoparticles, which was supported by package of fluorescent dyes.40 Sun et al. stated that reducing the molecular weight of PEG would enhance cellular uptake of PEG-coated Fe3O4 nanoparticles, as evidenced by inductively coupled plasma-atomic emission spectrometry.41 Although quantification of PEG in animals can be realized through isotope


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labeling technique,30,42 it is still desirable to develop an alternative method to determine the amount of PEG derivatives, because labeling or tagging PEGylated nanoparticles with dyes, isotope or metal NPs, often leads to significant changes in the physical-chemical properties, like size, stiffness and surface groups. In other words, the labeled or tagged PEGylated NPs are not the original ones for accurate analysis. Therefore, it is imperative to establish a direct method to quantify cellular uptake and drug release of PEGylated NPs without tags. In recent years, liquid chromatography tandem mass spectrometry (LC-MS/MS) has been widely used in quantification of therapeutic drugs, including PEGylated protein drugs.43-47 Gong and his co-workers used LC-MS/MS coupled with in-source collision-induced dissociation to quantify PEG and PEGylated proteins.48 Thus, along this line, we hypothesize that PEG and PEGylated drug without tags also can be quantified exactly via LC-MS/MS with in-quadrupole collision-induced dissociation(CID). However, to the best of our knowledge, no work about quantifying the cellular uptake of PEGylated drugs and released drugs in vitro and in vivo via LC-MS/MS with in-quadrupole CID is reported. When cells are incubated with PEGylated nanomedicines, they are internalized and followed by releasing drugs in cells to fulfill their therapeutic task. However, how PEGylation affects interactions between nanomaterials and biosystem. How many of the PEG-conjugated drugs and released drugs are in cells and organs with time. Last but not least, what the rate of drug release is in the actual cellular environment and organs. We expect to clear these clouds away through several ways, especially LC-MS/MS.


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Scheme 1. The formation of PEG-AM-Dox/PEG-HZ-Dox NPs and quantification of cellular uptake and released Dox by various methods.

In this work, we took PEGylated doxorubin nanoparticles (PEG-Dox NPs) as model to quantify the cellular uptake of PEG-Dox and released Dox from PEG-Dox NPs in actual cellular environment and in vivo through various methods (Scheme 1). The stable urethane and acid-


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labile hydrazone bonds were constructed between PEG and Dox to examine the possibility of different drug release rates. Furthermore, PEG with various molecular weights was used for studying the effect of PEG length on biological activity. EXPERIMENTAL SECTION Materials: Polyethylene glycol monomethyl ether 400 (PEG400) was purchased from TCI. Polyethylene glycol monomethylether, 550 (PEG550) was purchased from Alfa Aesar. Poly(ethylene glycol methyl ether), average M.W. 750 (PEG750) and 4-nitrophenyl chloroformate (NPC) were purchased from Acros. Poly(ethylene glycol) methyl ether average Mn 2000 (PEG2K), poly(ethylene glycol) methyl ether average Mn 10000 (PEG10K) and poly(ethylene glycol) methyl ether average Mn 20000 (PEG20K) were purchased from Aldrich. Hydrazine hydrate was purchased from Tianjin fuchen chemical reagents factory. Triethylamine was purchased by Aladdin. Doxorubicin.HCl (Dox.HCl) was purchased from HISUN (Zhejiang, China). The synthesis of PEG-Dox NPs was presented in Supporting Information. Cellular uptake and Cell viability assays and. The detailed data were presented in Supporting Information. Flow cytometry: The HeLa cells were seeded in six-well culture plates at a density of 5 × 105 cells per well and allowed to adhere for 24 h. After that, the cells were treated with PEG-Dox NPs (8.62 nmol mL-1, 1mL) for 5 h at 37 °C. Thereafter, the culture medium was removed, and the cells were washed with PBS three times and treated with trypsin. Then, 1.0 mL of PBS was added to each culture well, and the solutions were centrifuged for 5 min at 1000 rpm. After the removal of the supernatants, the cells were resuspended in 1 mL of PBS. Data for the 10,000 gated events were collected, and analyses were performed by flow cytometry (Beckman, California).


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LC-MS/MS analysis of Dox, PEG550-AMDox NPs, PEG550-HZ-Dox NPs and PEG20KAM-Dox NPs in HeLa cells: The HeLa cells were seeds in 10 cm cell culture dish at a density of 1 × 106 cells and allowed to adhere overnight. Then the cells were treated with PEG-Dox NPs (8.62 nmol mL-1, 7mL) for 1 h and 6 h at 37 oC, respectively. Thereafter, the culture medium was removed, and the cells were washed with PBS three times and treated with trypsin. The amount of cells was 2×106 by cell counting. Then ultrapure water (1mL) was added to collect cells, and cells were disrupted by Ultrasonic Cell Disruption System. After adding DMSO (1 mL), the mixture was centrifuged for 5 min at 10000 rpm. The supernatant was used for analysis and stored at – 80 oC. Extraction of cell nuclei experiment: The experiment was done according to the manual of KeyGEN nuclei isolation Kit (KGA826) which was purchased from KeyGEN BioTECH. Tumor inhabitation in vivo. The mice were injected with Dox or PEG-Dox NPs (equivalent Dox: 5 mg kg-1) intravenously. Mice were divided into 6 groups (n =10): (1) mice (Con) treated with saline; (2) Dox; (3) PEG550-AM-Dox NPs; (4) PEG2K-AM-Dox NPs; (5) PEG2K-HZDox NPs and (6) PEG10K-AM-Dox NPs. The mice were injected with Dox or PEG-Dox NPs (equivalent Dox: 5 mg kg-1) intravenously. These processes were carried out every 48 hours for three times. Tumor volume and body weight were measured every 2 days.

RESULTS AND DISCUSSION The synthetic process of PEG-Dox was shown in Scheme S1 (supporting information). The similar structure has been reported in previous literature.49-51 The PEG-Dox conjugates included PEG-AM-Dox and PEG-HZ-Dox, where the linkages are –OC(=O)NH– and –OC(=O)NH-N=C–,


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respectively. The PEG chain possesses an average molecular weight of 400, 550, 750, 2K, 10K, and 20K, respectively. Therefore, they were coded generally as PEG-Dox, and particularly as PEG550-AM-Dox or PEG10k-HZ-Dox, etc. The typical peaks of PEG (3.2-3.6 ppm) and Dox (7.5-8.0 ppm) could be found in proton nuclear magnetic resonance spectrum of PEG400-AMDox (Figure S1A), indicating the successful conjugation of PEG with Dox. All the PEG-Dox could self-assemble into stable nanoparticles. Briefly, a DMF solution of PEG-Dox or PEG-HZDox was added into distilled water. After stirred for 4 h, the mixture was dialyzed against distilled water with a cellulose membrane for 3 days. The spherical nanoparticles were showed in Figure S1B, C and S2, and the diameter observed by transmission electron microscopy (TEM, diameter 10-100 nm) was smaller than that measured by dynamic light scattering (DLS, diameter 40-150 nm) (Figure S1D and Table S1). The optical properties were recorded in Figure S1E and S3. Compared to that of pure Dox, the red shift of the absorption band of PEG-Dox and the absorption tails extending into the long wavelength region indicate formation of NPs in water because of the Mie effect of nanoparticles.52 No fluorescence was observed in Figure S1F (left) and S4 for PEG-Dox NPs in water due to aggregation-caused quenching (ACQ).53,54 PEG-Dox could dissolve in DMF, which led to disappearance of ACQ and emit fluorescence as shown in Figure S1F (right) and S4.


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Figure 1. Representative CLSM images of HeLa cells incubated with A) PEG550-AM-Dox NPs, B) PEG10K-AM-Dox NPs, C) PEG550-HZ-Dox NPs and D) PEG10K-HZ-Dox NPs for 1, 6, 12


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h. For each panel, the images from left to right show cell nuclei stained by Hoechst 33258 (blue), PEG-Dox and PEG-HZ-Dox fluorescence in cells (red), and overlays of both images. Scale bar, 50 µm. E, F and G) The trend of fluorescent intensity in a cell along the direction of the arrows marked in C).

The cellular uptake in human cervical carcinoma (HeLa) cells was evaluated by confocal laser scanning microscopy (CLSM). The cell nuclei were stained with Hoechst 33258 (blue). Red fluorescence of the dissociated PEG-Dox or released Dox was used as a measurement of the cell up-take, dissociation of the NPs and Dox released. CLSM images of the four samples (PEG550AM-Dox (A), PEG10k-AM-Dox (B), PEG550-HZ-Dox (C), and PEG10k-HZ-Dox (D)) were collected in Figure 1 and the others were shown in Figure S5 and S6. For all samples examined, the red fluorescence within the cells was weak at 1 h, and became stronger at 6 h and 12 h. The enhanced red fluorescence with the incubation time from 1 to 12 h in Figure 1 showed that NPs could be endocytosed by cells and disintegrated in cells to emit fluorescence. Otherwise, the red fluorescence from the NPs could hardly be observed because of ACQ (Figure S1F). Red fluorescence was poor in cell nuclei in 1 h (Figure 1E and S7A), and distributed obviously in cell nuclei and cytoplasm after 12 h. The intensive red fluorescence in cell nuclei (Figure 1E-G and S7) validated that Dox could be released and located in nuclei. To compare the fluorescent intensity of low molecular weight PEGylated Dox NPs (LPEGDox NPs, average molecular weight of PEG: 400, 550 and 750) and high molecular weight PEGbonded Dox NPs (HPEG-Dox NPs, average molecular weight of PEG: 10K and 20K) in cells, we chose PEG550-Dox NPs and PEG10K-Dox NPs as examples in Figure 1. Cells cultured by PEG10K-Dox NPs in Figure 1B and 1D (PEG10k) showed much weaker red fluorescence than


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that by PEG550-Dox NPs in Figure 1A and 1C, indicating that the molecular length of PEG has significant influence on the cellular uptake of the NPs. HPEG led to enhanced hydrophilicity of the NPs, lower affinity of the NP surface with the cell membrane, and less efficient endocytosis. The PEG-Dox used in Figure 1A and 1B possess a linkage of –OC(=O)NH– between PEG and Dox, while those in Figure 1C and 1D had a linkage of –OC(=O)NH-N=C–. It is well known that the latter is more facile to acidic hydrolysis. Unfortunately, Figures 1A and 1C did not show obvious difference in red fluorescence, and Figures 1B and 1D did neither. The possible explanation is that (1) these two linkages do neither influence cellular uptake or disassociation of the NPs; (2) The CLSM cannot differentiate dissociated PEG-Dox and released Dox.

Figure 2. Flow cytometry of HeLa cells incubated with PEG550-AM-Dox NPs, PEG550-HZDox NPs, PEG20K-AM-Dox NPs and PEG20K-HZ-Dox NPs for 5 h.


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Furthermore, flow cytometry (FCM) was used to compare quantitatively the fluorescence intensity in cells (Figure 2), which validated that the fluorescence intensity in cells incubated with PEG-HZ-Dox NPs was higher than that with PEG-AM-Dox NPs. The order of fluorescence intensity was PEG550-HZ-Dox NPs (50.9 %) > PEG550-AM-Dox NPs (33.1 %) > PEG20KHZ-Dox NPs (22.1 %) > PEG20K-AM-Dox NPs (14.5 %). There might be three reasons for this result. At first, HPEG-Dox NPs were less endocytosed than LPEG-Dox NPs. Second, HPEGDox NPs were more stable in living cells than LPEG-Dox NPs, which resulted in that PEG20KDox NPs were slowly decomposed to emit fluorescence. The last, PEG-HZ-Dox NPs were disintegrated in cells faster than PEG-AM-Dox NPs because of the acid-responsive hydrazone linkage. FCM was a wonderful method to quantify the fluorescence intensity in cells. However, there were similar problems in both CLSM and FCM. Was the enhanced fluorescence ascribed to continuous cellular uptake of NPs, or disassembly of NPs to recover the fluorescence, or the both? How many were NPs internalized as time went? How much Dox was released from NPs with prolonging incubation time? The cellular uptake of PEGylated Dox evaluated by FCM was reported by some work.50, 55 But the data from FCM could not precisely reflect the disassembly of PEG-Dox NPs and release of Dox. Therefore, more powerful and accurate analytical methods are needed.


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Figure 3. Cell viability of HeLa cells incubated with (A) Dox.HCl, (B) PEG550-AM-Dox NPs and PEG550-HZ-Dox NPs and (C) PEG20K-AM-Dox NPs and PEG20K-HZ-Dox for 24, 48, 72 h. *** P < 0.001. Relative to CLSM and FCM analysis, cytotoxicity is more directly related to released Dox from the PEG-Dox NPs. The cytotoxicities toward HeLa cells were evaluated by 3-(4,5dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assays. With increasing incubation time and Dox concentration, both Dox and PEGylated Dox NPs indicate enhanced cytotoxicity (Figure 3 and S8). PEG-Dox NPs showed lower cytotoxicity than free Dox because of the slow release of Dox from nanoscale formulations. About 80 % of cells were dead after incubated with Dox of 15 µg mL-1 for 24 h in Figure 3A. However, PEG-Dox NPs did not exhibit obvious cell inhibition effects at 24 h even with 15 µg mL-1 of equivalent Dox. The IC50 (half maximal inhibitory concentration) values were calculated from the MTT data and collected in Table 1. Because the action mechanism of Dox is combination with cell DNA, IC50 can reflect the total effect of endocytosis, dissociation, and Dox release. As shown in Figure 3 and Table 1, LPEG-Dox NPs were more toxic than corresponding HPEG-Dox NPs no matter the linkage was –OC(=O)NH– or –OC(=O)NH-N=C–, in agreement with results of cell uptake in Figure 1 and


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S5. It was also noticed that IC50 values of PEG-HZ-Dox were lower than that of PEG-AM-Dox. It is desired and imperative to further explaining the results from cytotoxicity. Table 1. IC50 of Dox, PEG-AM-Dox NPs and PEG-HZ-Dox NPs Sample

IC50 (nmol mL-1)

Sample

IC50 (nmol mL-1)

24 h

48 h

72 h

24 h 48 h 72 h

Dox

1.60

0.60

28

4.87

2.95

PEG400-HZ-Dox NPs

>28

6.88 2.77

PEG550-AM-Dox NPs

>28

10.49

4.96

PEG550-HZ-Dox NPs

>28

4.89 2.12

PEG750-AM-Dox NPs

>28

8.24

2.82

PEG750-HZ-Dox NPs

>28

5.21 1.80

PEG2K-AM Dox NPs

>28

21.14

6.67

PEG2K-HZ-Dox NPs

>28

8.33

4.6

PEG10K-AM-Dox NPs >28

>28

21.29 PEG10K-HZ-Dox NPs >28

>28

7.96

PEG20k-AM-Dox NPs

>28

18.43 PEG20K-HZ-Dox NPs >28

>28

9.19

>28

It is of pivotal importance to monitor accurately content of PEG-Dox and released Dox from PEG-Dox NPs. Herein, it is possible to use LC-MS/MS coupled with In-Quadrupole CID for quantifying PEG and PEGylated Dox according to our previous work.56,57 PEG and PEGylated Dox underwent dissociation in the second Quadrupole of mass spectrometer to generate a series of high resolution PEG-specific ions at m/z 133.08, 177.11, 221.13, 265.16, 309.18, 353.21, 397.23, which were corresponded to 3, 4, 5, 6, 7, 8, and 9 -CH2CH2O- units [(M+1)+1], respectively, as shown in Figure S9 and S10. The ion peak at m/z 133.08 was stable and highintensitive, and was chosen for quantitative analysis of PEG-Dox. PEG550, PEG550-NPC and PEG550-HZ possess similar HPLC retention time around 2.1−2.4 min (Figure S11). More importantly, PEG (Figure S9A and S11), Dox (Figure S9C) and PEG-Dox (Figure S9B and S10) had different retention time on HPLC. Therefore, it was possible to simultaneously quantify Dox and PEG-Dox in cells. The standard curves of PEG550-AM-Dox (Figure S7D), Dox, PEG550-


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HZ-Dox and PEG20K-AM-Dox (Figure S12) were obtained for quantitative analysis. Inquadrupole-CID LC-MS/MS was used to analyze the cancer cell samples. The cells were treated respectively with PEG550-AM-Dox NPs, PEG550-HZ-Dox NPs and PEG20K-Dox NPs with equivalent Dox for 1 h and 6 h at 37 oC. The content of Dox and PEG-Dox in HeLa cells was quantified according to the standard curves in Figure S13-16.


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Figure 4. A) The molar amount of PEG20K-AM-Dox, PEG550-AM-Dox and PEG550-HZ-Dox which were internalized by HeLa cells (amount: 2*10^6). B) The molar amount of Dox released from PEG20K-AM-Dox NPs, PEG550-AM-Dox NPs and PEG550-HZ-Dox NPs after they were internalized and metabolized by HeLa cells (amount: 2*10^6). **P < 0.01, *** P < 0.001 and n.s. P >0.05.


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We calculated the content of Dox and PEG-Dox as showed in Figure 4 and Table S2. The molar amount of PEG550-AM-Dox and PEG550-HZ-Dox were about 6 times than that of PEG20K-AM-Dox in HeLa cells although they were incubated under the same conditions (8.62 nmol mL-1of Dox, 6 h), indicating that high molecular weight PEG hindered endocytosis of NPs severely. As incubation time extended from 1 to 6 h, PEG550-AM-Dox was internalized into HeLa cells from 4.14 to 5.55 nmol. As shown in Figure 4B, released Dox was present in the HeLa cells after incubation with PEG-Dox NPs, and the molar amount of the released Dox was dependent on incubation time (PEG550-AM-Dox, 1 h vs. 6 h), PEG length (PEG10K-AM-Dox vs. PEG550-AM-Dox, 6 h) and linkage groups (PEG550-AM-Dox vs. PEG550-HZ-Dox, 6 h). More Dox was released from PEG550-AM-Dox with the increasing incubation time, which showed Dox could be released incessantly from PEGylated NPs. Dox released from PEG20KAM-Dox NPs was the least because of relatively poor internalization of PEG20K-AM-Dox NPs. More importantly, Dox released from PEG550-HZ-Dox NPs was more than that from PEG550AM-Dox NPs, which confirmed that acid-labile hydrazone bonds accelerated Dox release from NPs. There was no difference in the molar amount of PEG550-AM-Dox and PEG550-HZ-Dox in Figure 4A, but amount of Dox released is different in Figure 4B. We summed Dox and PEG-Dox for evaluating the cellular uptake of NPs in Figure S17, which demonstrated that molecular structure had little effect on cellular uptake. For PEG-Dox NPs, Dox was aggregated to form the core of the NP, while PEG formed the shell, and covered the effect of molecular structure to cellular uptake.


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Figure 5. Dox in cell nuclei and cytoplasm at different incubation time. HeLa cells (amount: 5*10^6) were incubated with PEG550-AM-Dox NPs (8.52 nmol mL-1, 7 mL).

It is well known that the action site of Dox is in cell nuclei. Complexing of Dox with DNA chains leads to inhibition of cell proliferation, and finally induces cell apoptosis. Herein, the Dox amounts in cell nuclei and cytoplasm were further determined by in-quadrupole-CID LC-MS/MS. The amount of Dox increased about 3 times in cell nuclei and about 4.6 times in cytoplasm from 1 h to 6 h as shown in Figure 5. It implied that Dox released from NPs could enter cell nuclei with enough time. It was also noticed that the amount of Dox in cytoplasm was about 65 times larger than that in cell nuclei at 6 h. This is because PEG-Dox NPs are usually internalized by cells through endocytosis and trapped in endosomes and lysosomes for a certain time. Only after


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endosome escape, release Dox could diffuse into cell nuclei.58 The fluorescence measurement in Figure S18 qualitatively supported the above results. The similar data were gotten when cells were incubated with PEG550-HZ-Dox NPs as shown in Figure S19, which demonstrated that there were more released Dox in cytoplasm than cell nuclei in 6 h.

Figure 6. A) The tumor volume of mice for 15 days. B) The body weight of mice for 15 days. To evaluate the effect of PEG with various molecular weights and different linkage bonds on the antitumor activity, biodistribution and drug release in vivo, we chose four PEG-Dox NPs (PEG550-AM-Dox, PEG2K-AM-Dox, PEG2K-HZ-Dox and PEG10K-AM-Dox NPs), using free Dox as control. It was surprising that Dox showed stronger antitumor effect than PEG-Dox NPs. When mice were treated intravenously with Dox, the tumor volume was smallest among all of the experimental groups in Figure 6A. Then PEG2K-HZ-Dox NPs showed modest antitumor effect but PEG(550, 2K, 10K)-AM-Dox NPs had no effect. However, Dox indicated serious side effects, which made the weight of mice drop sharply as shown in Figure 6B. Furthermore, two mice treated with Dox were dead while no mice died in other groups. At last, the main organs and tumors were isolated ex vivo. The photo of tumors in Figure S20A and the weight of tumors


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in Figure S20B also confirmed that the order of the antitumor effect was Dox > PEG2K-HZ-Dox NPs > PEG (550, 2K, 10K)-AM-Dox NPs. In order to reveal the nature for tumor inhibition, we investigated the biodistribution and drug release in detail by FLIV and LC-MS/MS.

Figure 7. Fluorescence intensity of main organs Mice were treated with A) Dox, B) PEG550AM-Dox NPs and C) PEG10K-AM-Dox NPs. B (brain), H (heart), Li (liver), S (spleen), Lu (lung), K (kidney), G (female genitalia) and T (tumor). FLIV is an available method for monitoring drug delivery. However, the excitation and emission wavelengths of Dox are too short to be detected in vivo by fluorescence imaging system as shown in Figure S21. The brain (B), heart (H), liver (Li), spleen (S), lung (Lu), kidney (K), female genitalia (G) and tumor (T) were imaged ex vivo. The fluorescence concentrated in B, H, Li, K, G and T, while little fluorescence was seen in S and Lu in Figure S22, Figure S23A and C. It seemed that all NPs and Dox showed the similar biodistribution in mice. We quantified the average fluorescence intensities in Figure 7, Figure S23B and D, which showed similar poor fluorescence in S and Lu once again. Fluorescence imaging only gave limited information about biodistribution, which could not explain the curative effect and the toxicity. We also did not know the fluorescence was from Dox or PEG-Dox, and might get the wrong results from fluorescence imaging in vivo for monitoring drug delivery.


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LC-MS/MS gave us thorough data to monitor drug delivery and release. The concentration of Dox in tumor was highest in all groups when mice were treated with Dox in Figure 8. The concentration of Dox released from PEG2K-HZ-Dox NPs in tumor kept about 25 ng g-1 for 46 h. However, other PEG-Dox NPs released about 20 ng g-1 of Dox, and amount of Dox decreased rapidly in 46 h as shown in Figure 8A. The main reason for curative effect was ascribed to the different concentration of Dox in tumor: Dox > PEG2K-HZ-Dox NPs > PEG-AM-Dox NPs. More importantly, we calculated the concentration rate of Dox and PEG-Dox on the behalf of release effiency in Figure 8B. The rate was about 0.075 for PEG2K-HZ-Dox NPs, but it was below 0.025 for PEG(550, 2K, 10K)-AM-Dox NPs, further confirming acid-sensitive hydrazone bond was effective for releasing Dox in tumor. Dox bring severe side effect, especially cardiotoxocity.59 There was much Dox concentrated in H as shown in Figure S24, which led to indiscriminate damage. Then Dox in organs and tumor decreased as time went by due to metabolism. However, as shown in Figure S25, the amount of Dox released from PEG-Dox NPs was less than 50 ng g-1 and the rate of Dox and PEG-Dox was below 0.025 except PEG2K-HZDox NPs, which meant –OC(=O)NH– was pretty stable even in vivo, including complicated tumor microenvironment.


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Figure 8. A) The concentration of Dox in tumor by LC-MS/MS when mice were treated with Dox and PEG-Dox NPs. B) the concentration rate of Dox and PEG-Dox in tumor. CONCLUSION In this report, we systematically investigated the cellular uptake and drug release of PEGylated nanomedicines. Enhanced fluorescence was observed for HeLa cells incubated with PEG-Dox NPs with low molecular weight of PEG and acid-responsive linker, as evidenced by CLSM and FCM. However, CLSM and FCM could not distinguish the cellular uptake or disassociation of PEG-Dox NPs, and differentiate fluorescence from PEG-Dox or released Dox. MTT assays revealed that PEGylated Dox NPs with an acid-sensitive linkage and low molecular weight PEG possess potent cytotoxicity to HeLa cells. LC-MS/MS was used to precisely quantify the PEGDox and released Dox, and validated the better cellular uptake of PEG-Dox NPs with low molecular weight of PEG and faster drug release of PEG-Dox NPs with acid-labile hydrazone bonds. The relationship of antitumor activity and drug content in tissues, organs and tumor were further disclosed by LC-MS/MS. Free Dox showed the strongest antitumor effect because the


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concentration of Dox in tumor was highest among those groups treated with Dox and PEG-Dox NPs. There was little Dox released from PEG-AM-Dox NPs in whole body levels, which result in the poor tumor inhibition. Interestingly, acid-sensitive hydrozone bonds were beneficial for Dox release so PEG2K-HZ-Dox NPs showed suboptimal antitumor effect. The precise quantification of PEGylated nanomedicines and released drug in living cells gave us a new horizon to monitor the cellular uptake and release behavior of PEGylated NPs through LCMS/MS. This work provides a strong guiding significance for the evaluation the preclinical effect of the PEGylated nanomedicines. ASSOCIATED CONTENT Supporting Information. Experimental section; the physical and chemical properties of PEGDox NPs; CLSM images of PEG-Dox NPs; cytotoxicity; chromatograms from LC-MS/MS; the photo of tumors ex vivo and tumor weight; fluorescence imaging in vivo (Scheme S1, Figure S1S25, Table S1 and S2). This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *Zhigang Xie E-mail: [email protected] *Jingkai Gu E-mail: [email protected]


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ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (Project No. 51522307, 81673396, 81430087, 81603182 and 81673502).

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For Table of Contents Use Only The Effect of Molecular Structure on Cytotoxicity and Antitumor Activity of Pegylated Nanomedicines Wenhai Lin, Lei Yin, Tingting Sun, Tingting Wang, Zhigang Xie, Jingkai Gu and Xiabin Jing


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The Effect of Molecular Structure on Cytotoxicity and Antitumor Activity

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