Dual pH-Responsive Shell-Cleavable Polycarbonate Micellar

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Biological and Medical Applications of Materials and Interfaces

Dual pH-responsive Shell-cleavable Polycarbonate Micellar Nanoparticles for In Vivo Anticancer Drug Delivery Shaoqiong Liu, Robert Ono, Chuan Yang, Shujun Gao, Jordan Tan, James L Hedrick, and Yi Yan Yang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b01954 • Publication Date (Web): 14 May 2018 Downloaded from http://pubs.acs.org on May 14, 2018

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ACS Applied Materials & Interfaces

Dual pH-responsive Shell-cleavable Polycarbonate Micellar Nanoparticles for In Vivo Anticancer Drug Delivery Shaoqiong Liu1, Robert Ono2, Chuan Yang1, Shujun Gao1, Jordan Yong Ming Tan1, James L. Hedrick2*, Yi Yan Yang1* 1

Institute of Bioengineering and Nanotechnology, 31 Biopolis Way, The Nanos, #04-01, Singapore 138669 Phone: (+65-6824-7106); E-mail: ([email protected]) 2 IBM Almaden Research Center, 650 Harry Road, San Jose, California 95120, USA Phone: (+1-408-927-1632); E-mail: ([email protected])

Keywords: pH responsive, shell cleavable micelles, bortezomib, catechol-functionalized polycarbonate, boronate ester bond, in vivo antitumor activity

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Abstract

To exploit tumor and intracellular microenvironments, pH-responsive diblock copolymers of PEG and catechol-functionalized polycarbonate with acid-labile acetal bond as the linker are synthesized to prepare micellar nanoparticles that shed the shell at acidic tumor tissues and inside cancer cells, hence accelerating drug release at the target. The pH-dependent cleavage of the shell is demonstrated at pH 5.0 and 6.5 using GPC and 1H NMR. Bortezomib (BTZ, an anticancer drug containing a phenylboronic acid group) is conjugated to the polymers through formation of pH-responsive boronate ester bond between boronic acid and cetachol in the polymers. Dual pH-responsive bortezomib-polymer conjugates (BTZ-PC) self-assemble into micellar nanoparticles of small size ( 0.05; d: BTZ-PC2 vs. BTZ-PC4 at pH 6.5, p < 0.01.

One key design of the micelles is to shed the shell inside the tumor tissues and the cancer cells, hence accelerating drug release and enhancing cellular uptake/intracellular killing at the target. To evaluate whether BTZ-PC2 can be more efficiently internalized by cancer cells at pH 6.5, cellular uptake of the dual pH-sensitive BTZ-PC2 was studied and compared between pH 7.4 19 ACS Paragon Plus Environment


and pH 6.5. As shown in Figure 4B, intracellular BTZ content in BT-474 cells that were treated with BTZ-PC2 was much higher than that in the cells that were incubated with free BTZ (p < 0.05), demonstrating that the micelles entered the cancer cells with higher efficiency. Moreover, the internalization of BTZ-PC2 micelles was significantly enhanced at pH 6.5 as compared to that at pH 7.4 (p < 0.05). To study if PEG shell cleavage at pH 6.5 affects the intracellular uptake of BTZ, an analogous polymer PC4 was synthesized with a similar structure as PC2 except that the linker between PEG and polycarbonate is a noncleavable carbonate bond instead of the acetal junction in PC2. BTZ-loaded micelles were self-assembled from BTZ-PC4 conjugate with similar size (denoted as BTZ-PC4, Table 1). Figure 4B shows that there was no effect of pH on internalization of BTZ when the single pHresponsive BTZ-PC4 micelles were used (p > 0.1). At pH 7.4, the uptake of BTZ by the cells that were treated with BTZ-PC4 was comparable to that of BTZ-PC2 (p > 0.05). At pH 6.5, however, a significantly increased BTZ content was observed inside the cells that were incubated with BTZ-PC2 (p < 0.01). Taken together, the results demonstrated that BTZ-PC2 exhibited significantly enhanced cellular internalization at pH 6.5. Similar results were also reported by Wang et al., where acidic pH triggered the cleavage of PEG corona and led to exposure of hydrophobic core, which promoted cellular uptake 28, 46.

The pH-responsive cytotoxicity of the dual pH-sensitive BTZ-PC2 micelles and the single pH-sensitive BTZ-PC4 micelles was investigated on human breast cancer MDA-MB-231 and BT-474 cell lines (Figure 5). The cells were cultured in the medium containing BTZ-PC2 or BTZ-PC4 at pH 7.4 or 6.5, with free BTZ treatment as the control. In both cell lines, free BTZ had a similar IC50 value (drug concentration that inhibits cell growth by 50%) at pH 6.5 and pH 7.4 (Figure 5). However, when the cancer cells received BTZ-PC2 or BTZ-PC4 treatment, IC50 value of BTZ at pH 6.5 was significantly lower than that at pH 7.4 (p < 0.05), demonstrating that both dual pH-responsive BTZ-PC2 micelles and single pH-responsive 20 ACS Paragon Plus Environment

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BTZ-PC4 micelles exhibited greater cytotoxicity to the cells under the acidic condition. The enhanced cytotoxicity of BTZ-PC2 and BTZ-PC4 at pH 6.5 might be atttributed to faster BTZ release (Figure 3) and greater cellular uptake of BTZ (Figure 4B). Furthermore, BTZ-PC2 and BTZ-PC4 showed comparable cell growth inhibition at pH 7.4 (Figure 5). However, at pH 6.5, BTZ-PC2 showed significantly higher in vitro cytotoxicity than BTZ-PC4 ( p < 0.05) possibly due to the increased cellular uptake of BTZ when delivered by BTZ-PC2 (Figure 4B). These results demonstrated that the dual pH-sensitive BTZ-PC2 micelles exerted significantly enhanced cytotoxicity in the acidic environment than the single pH-responsive BTZ-PC4 micelles. It is worth noting that no significant cytotoxicity was induced by the blank polymers up to 2000 µg/mL as seen in Figure S-3. Therefore, the observed anticancer effect of BTZPC2 and BTZ-PC4 was from BTZ. These findings indicated that dual pH-responsive BTZPC2 might exert enhanced anticancer effect in acidic tumor tissues.



0.040

(A)

pH 7.4

*

pH 6.5

** IC50 (µg/mL)

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b

b

c

0.020

c

a

0.000 BTZ

BTZ-PC2

BTZ-PC4

(B)

Figure 5. In vitro anticancer effect of BTZ, BTZ-PC2 and BTZ-PC4: IC50 values against MDA-MB-231(A) and BT-474 (B) cells after incubation with free BTZ, BTZ-PC2 and BTZPC4 for 48 h in medium with pH 7.4 or pH 6.5 at 37°C. (a: BTZ at pH 7.4 vs. BTZ at pH 6.5, p > 0.05; b: BTZ-PC2 vs. BTZ-PC4 at pH 7.4, p > 0.05; c: BTZ-PC2 vs. BTZ-PC4 at pH 6.5, p < 0.05; *: p < 0.05; **: p < 0.01)

To evaluate proteasome activity of BTZ-PC2, proteasome-GloTM Chymotrypsin assay (Promega) was performed on BT-474 cells after 24-h incubation with BTZ or BTZ-PC2 (BTZ concentration: 0.02 µg/mL). As shown in Figure 6A, free BTZ showed comparable proteasome activity at pH 7.4 and pH 6.5 (p > 0.05). However, at pH 6.5, BTZ-PC2 demonstrated significantly higher proteasome activity than at pH 7.4 (p < 0.05), which was


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attributed to enhanced cellular uptake of BTZ (Figure 4B) and accelerated BTZ release from the micelles at pH 6.5 (Figure 3).

(A)

(B)

Figure 6. Anticancer mechanism of BTZ-PC. (A) Proteasome activity of BT-474 cells after treatment with BTZ and BTZ-PC2 for 24 h (BTZ concentration: 0.02 µg/mL); (B) Apoptosis of BT-474 cells after treatment with BTZ and BTZ-PC2 for 24 h (BTZ concentration: 0.02 µg/mL). (a: p > 0.05, *: p < 0.05)



The apoptosis of BT-474 cells induced by BTZ or BTZ-PC2 treatment was evaluated by the Annexin V (AnV) assay using flow cytometry. The number of apoptotic cells was analyzed after 24-h treatment with different formulations at pH 7.4 or 6.5 (Figure 6B). Compared to the physiological condition (pH 7.4), BTZ treatment induced a similar number of apoptotic cells at pH 6.5 (p > 0.1). However, BTZ-PC2 treatment resulted in a significantly higher number of apoptotic cells at pH 6.5 than at pH 7.4 (p < 0.05). Moreover, the treatment with BTZ-PC2 at pH 6.5 induced cell apoptosis as effectively as free BTZ (p > 0.05). The results further demonstrated that BTZ was effectively released from BTZ-PC2 micelles in the acidic environment. More importantly, the released BTZ molecules remained bioactive as a proteasome inhibitor and induced cancer cell apoptosis.

The in vivo antitumor efficacy of BTZ-PC2 was investigated in nude mice bearing BT-474 xenografts. There was no significant difference among different groups in terms of tumor volume prior to various treatments. As shown in Figure 7A, at the end of the treatment, the mice treated with BTZ-PC2, free BTZ and saline had a mean tumor volume of 105 mm3, 150 mm3 and 260 mm3, respectively. The treatment with BTZ or BTZ-PC2 reduced tumor volume significantly as compared to saline control (BTZ, BTZ-PC2 vs. saline, p < 0.05). Notably, the BTZ-PC2 formulation completely inhibited tumour growth and outperformed free drug (BTZPC2 vs. BTZ, p < 0.05). The greater antitumor efficacy of BTZ-PC2 might be due to the accumulation of BTZ-PC2 in the tumor through the EPR effect, followed by shell cleavage and simultaneous release of free BTZ from the micelles triggered by the local acidic environment of tumor tissues and/or intracellular acidity29, 47.


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(A)

(B)

Figure 7. In vivo antitumor activity and body weight changes in nude mice bearing BT-474 xenografts after i.v. injection of free BTZ and BTZ-PC2 micelles. (a) Tumor growth as a function of time, mice injected with saline were used as control (* means p < 0.05, free BTZ or BTZ-PC2 vs. control; + means p < 0.05, BTZ-PC2 vs. free BTZ); (b) Changes of mouse body weight during the treatment. Mice injected with saline were used as control. (* means p < 0.05, free BTZ vs. control; + means p > 0.05, BTZ-PC2 vs. control; ξ means p < 0.05, BTZPC2 vs. free BTZ)



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Histological analysis of the tumors excised from the mice was performed. Compared to the control treated with saline, more apoptotic bodies (nuclei stained brown) were observed in the tumor of mice treated with free BTZ and BTZ-PC2 (Figure 8, BTZ or BTZ-PC2 vs. saline, p < 0.001). It is noteworthy that BTZ-PC2 induced a remarkably higher number of apoptotic cells than free BTZ in the tumor (Figure 8B, BTZ-PC2 vs. BTZ, p < 0.01). The results further demonstrated that BTZ-PC2 presented stronger antitumor activity in vivo and the antitumor mechanism was due to BTZ-induced apoptosis in cancer cells. (A) Control

BTZ

BTZ-PC2

(B)

Figure 8. (A) TUNEL staining of tumors at 12 days after treatment with BTZ and BTZ-PC2. Saline was used as control. Scale bar: 50 µm; (B) Quantification of TUNEL-positive cells in tumor sections. (* means p < 0.05, BTZ-PC2 vs. free BTZ; ** means p < 0.05, BTZ or BTZPC2 vs. control).

Changes in body weight with time were monitored to evaluate the general toxicity of free BTZ and BTZ-PC2 formulations (Figure 7B). The treatment with free BTZ caused significant 26 ACS Paragon Plus Environment

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body weight loss as compared to saline treatment. In contrast, there was no significant difference in the body weight between BTZ-PC2 and saline treatments (p > 0.05). The systemic toxicity of free BTZ and BTZ-PC2 was further evaluated by analysing hematoxylin and eosin (H&E)-stained major organs of the mice at the end of the treatment course. Treatment with BTZ or BTZ-PC2 did not cause obvious tissue damage such as structural degeneration of tissue necrosis in lung, spleen kidney and heart. However, free BTZ treatment induced significant hepatotoxicity as evidenced by cytoplasmic vacuolation (indicated by white arrows) and hydrop degeneration (indicated by white dotted area) (Figure 9 and Figure S-4 C and D). There was no significant difference in the H&E findings between the control and BTZ-PC2 formulation, where normal appearance of organized hepatocytes separated by sinusoidal capillaries were observed The results further demonstrated BTZ-PC2 safety at the therapeutic doses. This might be attributed to the fact that BTZ-PC2 delivered BTZ to the tumour more effectively than free BTZ through the EPR effect and the pH-triggered release at the tumour site, which reduced non-specific toxicity of free BTZ in the healthy liver tissue.



Figure 9. Haematoxylin & erosin (H&E) staining of major organs of mice including lung, spleen, kidney, heart and liver at 12 days post injection of BTZ and BTZ-PC2. Saline was used as control. White arrows denote cytoplasmic vacuolation; White dotted area denotes hydrop degeneration. Scale bar: 200 µm.

4. Conclusion The pH-responsive diblock copolymers of PEG and catechol-functionalized polycarbonate were successfully synthesized and used to prepare pH-sensitive shell-cleavable micellar nanoparticles. BTZ was conjugated to the polymers through pH-sensitive boronate ester bond between boronic acid group in BTZ and catechol group in the polymers. This approach allowed the micelles to load BTZ at high loading levels. The use of the dual pH-responsive BTZ-loaded micelles enhanced intracellular uptake of BTZ than that of the BTZ-loaded 28 ACS Paragon Plus Environment

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micelles without pH-sensitive linker between the shell and the core. At acidic pH, the dual pH-responsive micelles showed higher proteasome inhibition, stronger apoptotic activity and greater in vitro cytotoxicity as compared to pH 7.4 in normal tissues. More importantly, in the human breast cancer BT-474 xenograft mouse model, the dual pH-responsive BTZ-loaded micelles demonstrated a stronger antitumor effect than free BTZ while mitigating hepatotoxicity of BTZ. These dual pH-responsive shell-cleavable micellar nanoparticles can potentially be used to deliver BTZ for cancer therapy.

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Supporting Information.The synthesis and characterization of monomers and polymers, viability data of BT-474 cells after 48-h incubation with the blank polymers, and high magnification of H&E staining of liver sections are included in the Supporting Information.

Acknowledgements The authors would like to acknowledge the financial supports from the Institute of Bioengineering and Nanotechnology (Biomedical Research Council, Agency for Science, Technology and Research, Singapore), and IBM Almaden Research Center, USA.


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The table of contents

Keywords pH responsive, shell cleavable micelles, bortezomib, catechol-functionalized polycarbonate, boronate ester bond, in vivo antitumor activity

Shaoqiong Liu1, Robert Ono2, Chuan Yang1, Shujun Gao1, Jordan Yong Min Tan1, James L. Hedrick2*, Yi Yan Yang1*

Dual pH-responsive Shell-cleavable Polycarbonate Micellar Nanoparticles for In Vivo Anticancer Drug Delivery

Acidic pH

Dual pH triggered drug release

Dual pH-responsive bortezomib-loaded micelles (BTZ-PC) are synthesized through selfassembly of amphiphilic diblock copolymers of PEG and bortezomib-conjugated polycarbonate with pH-sensitive acetal junction. BTZ-PC micelles show high drug loading level and exhibit enhanced in vitro cytotoxicity in acidic environments. More importantly, the BTZ-PC micelles achieve a strong antitumor effect in a human breast cancer BT-474 xenigraft mouse model, while mitigating in vivo toxicity of free BTZ. These dual pHresponsive shell-cleavable micelles are a promising carrier for in vivo bortezomib delivery.


Dual pH-Responsive Shell-Cleavable Polycarbonate Micellar

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