Injectable and quadruple-functional hydrogel as an alternative

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Biological and Medical Applications of Materials and Interfaces

Injectable and quadruple-functional hydrogel as an alternative to intravenous delivery for enhanced tumor targeting Zhi-Qiang Zhang, Young-Min Kim, and Soo-Chang Song ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b10182 • Publication Date (Web): 02 Sep 2019 Downloaded from pubs.acs.org on September 2, 2019

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ACS Applied Materials & Interfaces

Injectable and Quadruple-Functional Hydrogel as an Alternative to Intravenous Delivery for Enhanced Tumor Targeting

Zhi-Qiang Zhang,† Young-Min Kim,† and Soo-Chang Song†,§, * †Center

for Biomaterials, Korea Institute of Science and Technology, Seoul 02792,

Republic of Korea § Department

of Biomolecular Science, University of Science and Technology (UST),

Daejeon 305-350, Republic of Korea

*Corresponding

author.

Email: [email protected]

ACS Paragon Plus Environment

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

ABSTRACT Intravenous (IV) route is most commonly used drug delivery approach. However, the targeting efficiency to tumor through IV delivery is usually less than 10%. To address this limitation, we report a new systemic delivery method utilizing injectable and quadruple-functional hydrogels to improve targeting efficiency through passive, active, and magnetic targeting, and hydrogel-controlled sustained release. The hydrogels consist of a folate/polyethylenimine-conjugated poly(organophosphazene) polymer, which encapsulates small interfering RNA (siRNA) and Au–Fe3O4 nanoparticles to form a nanocapsule (NC) structure by a simple mixing. The hydrogels are localized as a long-term ‘drug-release depot’ after a single subcutaneous injection and sol-gel phase transition. NCs released from the hydrogels enter the circulatory systems and then target the tumor through EPR/folate/magnetism triple targeting, over the course of circulation, itself prolonged by the controlled release. In vivo experiments show that 12% of NCs are successfully delivered to the tumor, which is a considerable improvement as compared to most results through IV delivery. The sustained targeting of gold to tumor enables two cycles of photothermal therapy, resulting in an enhanced silencing effect of siRNA and considerable reduction of tumor volume, which we are unable to achieve via simple intravenous injection.

KEYWORDS: Quadruple-functional hydrogel, Alternative to IV, Triple-targeting, Sustained release, Long-term therapy


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■ INTRODUCTION The leaky vasculature and defective lymphatic drainage of tumor tissues has long inspired researchers to passively target nanocarriers to tumors.1-3 However, the efficacy of passive targeting through enhanced permeability and retention (EPR) is hindered by several intrinsic and extrinsic factors of the tumor. These limiting factors include tumor heterogeneity, elevated interstitial fluid pressure, endosomal escape, and limitations of the nanocarriers themselves. Thus, the result to date has been low targeting efficiency. Alternatively, researchers have used active targeted delivery strategies, utilizing the binding between affinity ligands and overexpressed receptors on tumor cell surfaces.4-6 However, the targeting efficiency through conventional intravenous (IV) delivery is typically less than 10%.7 These low targeting results are primarily attributable to clearance by the mononuclear phagocytic system and insufficient EPR in human tumors.8-9 Active targeting is of little use if systemic delivery is unable to facilitate nanocarriers entry into the tumor microenvironment to take advantage of the increased affinity. Thus, efficient passive targeting is a prerequisite for nanocarriers designed to systemically target tumor.10 Magnetic targeting, which uses an exogenous magnetic field to target a tumor, can enhance penetration and accumulation of magnetic nanocarriers into tumors, thereby enhancing cellular uptake.11-13 Schleich et al. enhanced the targeting of paclitaxel to tumor by the combination of both active and magnetic strategies.13 However, the tumor volume increased more than 10-fold after at 15 days post-treatment. This low therapeutic result may be attributable to the short blood circulation time of



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nanocarriers after IV delivery, negating the benefits of magnetic targeting.14 Thus, an improved systemic drug delivery method to enhance targeting efficiency is highly desirable. As an alternative to IV delivery, subcutaneous (SC) administration using an injectable hydrogel may increase bioavailability, enable controlled drug release, and prolong the therapeutic window.15-16 Time-dependent hydrogel degradation and dissolution facilitates sustained drug release after a single SC injection,17-18 resulting in a prolonged circulation time. Use of hydrogels has additional advantages: 1) high drug-loading to reduce the number of injections, 2) requirement of only a small injection volume, and 3) the potential for controlled drug delivery, such as protein, chemicals, and gene.19-21 To address limitations of previous research, we propose a new systemic drug (e.g., siRNA)

delivery

method,

utilizing

folate-polyethylenimine

(PEI)-conjugated

poly(organophosphazene) (FPP)/siRNA/Au-Fe3O4 nanoparticle (FPP/S/N) assembled nanocapsule (NC) hydrogels. Our goal is to improve targeting efficiency via 1) EPR-mediated passive targeting, 2) folate-induced active targeting, 3) iron oxide nanoparticles-guided magnetic targeting, and 4) sustained delivery (prolonged circulation time). The gold in the nanoparticles facilities cyclic near-infrared (NIR) laser-induced photothermal therapy, itself rendered more effective due to long-term delivery and improved targeting. Thermal therapies, based on local temperature elevation within the tumor, can be used as a trigger for drug release22-23 or applied in combination with radiation and chemotherapy as an adjuvant cancer treatment.24-27


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The nanocarriers utilizing both gene delivery and photothermal therapy have been developed to explore their synergistic effect.28 Compared to cell membrane disruption induced by ablation (≥ 50 ºC), hyperthermia (42–45 ºC) facilitates cell uptake and transfection efficiency of the gene through enhanced cell membrane permeability and endosomal escape.29-30 Although researchers have studied multiple localized hyperthermia

and

single

ablation

for

cancer

treatment,24,

28

long-term

gene/photothermal therapy through one-time systemic delivery remained challenging. The principles of our drug delivery system are as follows. The NC hydrogels, formed by electrostatic interactions between the negatively charged siRNA and the polycations in PEI, protect the siRNA from endosomal entrapment and rapid enzymatic degradation. The Au–Fe3O4 nanoparticles are encapsulated by FPP via hydrophobic interactions. The iron oxide and gold are effective for magnetic targeting and NIR-induced hyperthermia, respectively (Figure 1A). After a single SC injection and subsequent sol-gel phase transition, the hydrogels are localized as a ‘drug-release depot’ (Figure 1B), from where the released NCs enter the lymphatic and blood circulatory system, and then effectively target the tumor via a triple-targeting strategy of EPR/folate/magnetism (Figure 1C). The tumor dies by a combination of long-term gene silencing and cyclic photothermal therapy (Figure 1D).



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Figure 1. Schematic illustration of long-term gene/photothermal combination therapy using a single SC injection of NC hydrogels. A) Assembly of FPP/S/N NCs. B) Sustained

release

of

NCs

from

the

hydrogels

after

SC

injection

and

temperature-responsive sol-gel phase transition. C) The released NCs were delivered through the lymphatic and blood circulation system and then targeted tumor via EPR/folate/magnetism triple-targeting effect. D) The tumor dies by a combination therapy.

■ RESULTS AND DISCUSSION Synthesis and characterization of NCs. With a view to realizing the triple targeted delivery of siRNA and Au-Fe3O4, we first synthesized Au-Fe3O4 nanoparticles and the FPP polymer. Mono-dispersed Au-Fe3O4 nanoparticles were synthesized though thermal decomposition and examined by transmission electron


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microscopy (TEM) and energy-dispersive X-ray spectroscopy (Figure S1A, B). Scanning TEM analysis revealed that Fe3O4 grew on the brighter Au seeds (Figure S1C). Within the nanoparticles, the Fe3O4 component acted as a magnetic force-assisted targeting moiety and the Au component served as thermal seeds under NIR laser irradiation to induce hyperthermia. The Au-Fe3O4 nanoparticles exhibit superparamagnetic properties at room temperature without remanence and coercivity in the M-H hysteresis (Figure S1D). The FPP polymer was synthesized by an amide coupling reaction between the primary amine groups of PEI and the terminal carboxyl groups of poly(organophosphazene) and folate, which facilitates the specific targeting of NCs to the folate receptor over-expressed cancer cells (Figure S2).31 Compared to the non-absorption of Fe3O4 and characteristic absorption of Au nanoparticles at 524 nm in aqueous solution, as observed in UV-visible spectra, an obvious red-shift of surface plasmon of pure Au once Au attached to Fe3O4 indicated that the Au-Fe3O4 could generate heat under NIR radiation (Figure S3). The interface communication between Au and Fe3O4 resulted in electron deficient electron population on Au, leading to the absorption shift to longer wavelength.32 The aggregation of Au-Fe3O4 by FPP encapsulation also enhances the red-shift.33 The rapid response of FPP/Au-Fe3O4 nanoparticles (FPP/N) solution without siRNA to an externally applied magnetic field suggested its potential for magnetic targeting (Figure S4). For triple-targeting and combination therapy, we then prepared FPP/S/N NCs, which were assembled by the physical mixing of FPP, Au-Fe3O4, and siRNA. TEM analysis of FPP/N solutions showed a core-shell nano-capsule structure, with Au-Fe3O4



encapsulated within the FPP polymer, formed due to hydrophobic interactions between the oleic acid of the nanoparticles and the L-isoleucine ethyl ester group of FPP (Figure 2A).24 The mean particle size decreased from 240 ± 42 nm to 145 ± 27 nm upon the addition of siRNA to form the NCs, due to interactions between positively charged FPP and negatively charged siRNA (Figure 2B). Supporting the TEM results, the particle size of the NCs, as quantified by dynamic light scattering (DLS), decreased when siRNA was mixed and incorporated within (Figure 2C). The negatively charged siRNA was neutralized step-by-step with increasing FPP contribution, suggesting that ionic interactions were the driving force behind NC formation (Figure 2D). The N/P ratio of 48 was selected to assemble the hydrogels in subsequent studies, as the NCs formed under these conditions were around 150 nm in size and possessed a slight positive charge, which should facilitate cellular uptake. Au-Fe3O4 nanoparticles induced hyperthermia was investigated as a function of Au-Fe3O4 concentrations and NIR laser power. The temperatures of FPP/N solutions were measured using a needle thermocouple under a NIR laser irradiation. With lower Au-Fe3O4 concentrations, the temperatures reached were outside of the hyperthermic range under a NIR laser power of 1.2 W/cm2, whereas temperatures between the mild and moderate hyperthermic range were achieved using 96 and 128 μg Fe/mL Au-Fe3O4, respectively (Figure 2E). Temperature increased to ablation range (≥ 50 ºC) when the power elevated to 1.6 W/cm2, whereas the temperature was too low at 0.8 W/cm2 (Figure 2F). Considering these results, hyperthermic conditions were at 1.2 W/cm2 NIR laser power and 128 μg Fe/mL Au-Fe3O4.


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Figure 2. Characterization of NCs. A) Representative TEM images of FPP/N NCs and B) FPP/S/N NCs solutions. The concentration of FPP was 0.5 wt% in water. C) The hydrodynamic particle size and D) zeta potential of the NCs with different N/P ratios. Temperature elevation in the FPP/N NC solutions depended on E) the concentration of Au-Fe3O4 and F) NIR laser powers.

Cytotoxicity and cellular uptake of NCs. The cytotoxicities of NCs and the combination with hyperthermia on MDA-MB-231 were measured by using



3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)assay (Figure 3A). The silencing of the polo-like-kinase (PLK-1) gene using siPLK-1 is selected because of its phase II clinical trials for solid tumors.34 No significant cell death was induced by FPP or FPP/N at concentrations of 5 mg FPP/mL and 125 μg Fe/mL compared to untreated cells. Two cycles of hyperthermia at 43 ºC alone was insufficient to kill cancer cells. Lipofectamine 2000 was used as a positive control to compare the transfection efficacy of siRNA with FPP. Cell viability decreased to 67% and 73% after treatment with siPLK-1 complex with lipofectamin 2000 and FPP with the assistance of magnetic attraction, respectively, suggesting that siRNA delivery using the FPP/S/N NCs was effective. The cell viability was further decreased when hyperthermia was applied and was related to the hyperthermic cycle number, indicating a synergistic effect between PLK-1 inhibition and hyperthermia. The live/dead and apoptosis/necrosis staining kits were used to confirm the enhanced cytotoxicity and cell death pathways by NIR laser-induced photothermal therapy (Figure 3B). The dead cells, stained red by ethidium homodimer-1 (EthD-1), were observed after treatment with both siPLK-1 and combination with hyperthermia. The quantity of dead cells correlated with hyperthermic cycle number, as confirmed by the MTT results. Correspondingly, only apoptotic cells, stained green using annexin V-cy3, were observed after treatment, whereas necrotic cells stained red using 7-aminoactinomycin-D (7-AAD), were not evident, suggesting that the combination therapy is safe. To explore the triple-targeting effect on cell uptake, cyanine5.5 (cy5.5)-tagged siPLK-1 was applied. Compared to the similar green fluorescence


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intensities by using lipofectamine 2000 or FPP, higher intensity was observed with additional magnetism, indicating that triple-targeting significantly enhanced cellular uptake of siRNA (Figure 3C). The gene silencing effect of siPLK-1, delivered by the different systems, to PLK-1 in MDA-MB-231 cells was examined by Western Blot analysis (Figure 3D). Compared to lipofectamine or dual targeting using FPP, significant down-regulation of PLK-1 was observed in the triple-targeting group. Combined, these results highlight the benefits of triple-targeting, with even better results achieved than gold standard lipofectamine.

Figure 3. In vitro evaluation of cytotoxicity and cellular uptake of FPP/S/N NCs. A)



Cytotoxicity induced by siPLK-1 and hyperthermia to MDA-MB-231 cancer cells. The symbol * indicates a statistical significance of P< 0.05, and ** indicates P< 0.01. B) Representative fluorescence microscopical images of stained cells using calcein AM/EthD-1 and annexin V-cy3/7-AAD and C) cellular uptake of cy5.5-tagged siPLK-1. Scale bar represents 100 μm. D) Western blot results of PLK-1 protein expression. β-actin was used as the loading control.

Sustained release of NCs from the hydrogels. The temperature-dependent phase transition of FPP hydrogels facilitates the sustained release of siRNA and Au-Fe3O4 nanoparticles following a single SC injection. The dissolution of the hydrogels is related to their viscosity at body temperature, which further influences the rate of drug release.22 The hydrogel viscosities and gelation temperature were adjusted by Au-Fe3O4 loading and the FPP concentration in aqueous solution (Figure S5). Compared to unloaded 6.5 wt% FPP, the viscosity at 37 ºC (198 Pa·s) did not significantly change after siRNA and nanoparticle loading. Although the gelation temperature decreased from 26.8ºC to 17.8 ºC, it is still good for injection. To examine the in vitro stability of NCs when released from the hydrogels, morphology and particle size distribution parameters were measured. TEM results of released NCs showed that the core-shell structure was maintained, with multiple Au-Fe3O4 nanoparticles encapsulated by FPP (Figure 4A). Meanwhile, the scanning TEM images highlight that the Au-Fe3O4 nanoparticles were released intact (Figure S6). DLS analysis showed a broad particle size distribution (PDI = 0.331), indicating that


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the dissolution of the hydrogels is a dynamic process, resulting in heterogeneous NCs release (Figure 4B). A mean particle size of 98 nm should facilitate the delivery of NCs through the blood and lymphatic circulation pathway, with the further targeting of tumor tissues.35 To further explore the in vivo release and delivery of siRNA and Au-Fe3O4 nanoparticles, NC hydrogels were subcutaneously injected to a tumor-xenograft model. The sustained release behavior of NC hydrogels was investigated by measurement of hydrogel weight loss and the corresponding release of siRNA and Au-Fe3O4 from the hydrogels, respectively (Figure 4C). Hydrogel dissolution was reflected by hydrogel weight loss. A sharp weight loss occurred within seven days and 32% of the hydrogel weight remained at 18 days post-injection, facilitating the fast release of the NCs in the early stages of therapy, which could be combined with cyclic photothermal therapy. The dissolution of the majority of the hydrogels was observed at 30 days post-SC injection, suggesting all the NCs were released when the hydrogels had been eliminated (Figure S7). Furthermore, release profiles of siRNA and Au-Fe3O4 from the hydrogels were both recorded. Au-Fe3O4 release trended with hydrogel weight loss, however, the release of siRNA was faster, with almost all the siRNA released within 18 days. The differential release rate can likely be attributed to the different driving forces that bind siRNA and Au-Fe3O4 to FPP, electronic and hydrophobic interactions, respectively. Real-time fluorescence imaging of NC hydrogels subcutaneously injected to tumor bearing mice was used to further examine the in vivo release of siRNA from



NC hydrogels. The fluorescence intensity of cy5.5-tagged siPLK-1 attenuated over time after a single SC injection, confirming the continuous release of NCs from the hydrogels (Figure 4D and 4E). Reductions in fluorescence from the hydrogels was converted to the release of siRNA against the initial injected dosage (Figure 4F), which correlated with the tendency of in vitro release of siRNA (Figure 4C). The results indicate that in vivo release and further tumor targeting of siRNA could be monitored by evaluation of cy5.5 intensity.

Figure 4. In vitro and in vivo sustained release of NCs from the hydrogels. A) In vitro representative TEM image and B) hydrodynamic particle distribution of the released NCs from the hydrogels cultured in PBS (pH = 7.4) at 37 ºC. C) In vitro weight loss of the hydrogels and corresponding release of Au-Fe3O4 and siRNA over time. D) In vivo fluorescence images of NC hydrogels upon SC injection. SiPLK-1 was labeled by cy5.5. E) In vivo fluorescence intensity quantification of cy5.5 remained in the hydrogels. F) In vivo release profile of siPLK-1 from the hydrogels against the initial amount of injection.


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We next assessed in vivo tumor targeting efficiency of released siRNA and Au-Fe3O4 nanoparticles from the hydrogels, respectively. The released NCs entered into the blood and lymphatic circulation system and were then delivered to the tumor site. We compared the effects of single EPR, dual EPR/folate, and triple EPR/folate/magnetism targeting on tumor targeting and gene silencing effects. Cognizant that IV injection is clinically recommended to deliver nanocarriers, NC solutions (2.6 wt% FPP) were administrated through IV injections to analyze the targeting efficiency of NC hydrogels (6.5 wt% FPP) against using SC injections. The same concentrations of siRNA and Au-Fe3O4 were applied in terms of both administrations. Real-time fluorescence images of the tumor were used to examine the targeting effect of cy5.5-tagged siPLK-1. The mice subjected to triple-targeting using the hydrogels exhibited the strongest fluorescence intensity than the other group mice over all time intervals (Figure 5A and 5B). The tumor targeting efficiency of siRNA was evaluated by comparing the quantitative results of cy5.5 intensity in the tumor against the injected dosage. The highest tumor targeting of siRNA was calculated as 10.2%

at

18

days

post-injection,

suggesting

both

triple-targeting

and

hydrogel-controlled sustained release are indispensable (Figure 5C). In contrast to sustained release using the hydrogels, the triple-targeting effect through IV injection was poor, as the extension time of NCs in the blood circulatory system was too short to result in efficient penetration to tumor under magnetic influence. Clearly, the extent of blood circulation time is key to enhancing tumor targeting efficiency.14 Meanwhile,



the effect of triple-targeting strategy was also confirmed by the presence of Au-Fe3O4 within the tumor tissues. Iron concentrations of Au-Fe3O4 within the tumor tissues were measured by atomic absorption spectroscopy (AAS). The targeting efficiencies of Au-Fe3O4 had reached 12.1%, which is a considerable improvement as compared to typical IV delivery (Figure 5C).7 The result was consistent with the targeting efficiency of siRNA. The reduction of siRNA targeting is possibly attributed to the time/light-dependent degradation of cy5.5 during the in vivo delivery course. The iron composition of Au-Fe3O4, stained as blue, was shown in the tumor section excised at 18 days, indicating successful tumor targeting of released Au-Fe3O4 (Figure S8). The main organs and tumor were excised with a view to imaging the bio-distribution of siRNA at 18 days post-injection (Figure 5D). Quantitative analysis confirmed that the tumor targeting efficiency using NC hydrogels and triple-targeting was about three-fold higher than the other groups (Figure 5E). Meanwhile, the fluorescence observed for healthy tissues were much lower than tumors, suggesting that SC administration using NC hydrogels is safe. These results suggest that the targeting efficiency of NCs to the tumor can be enhanced significantly through the triple-targeting strategy and sustained release of hydrogels, compared to one-shot release by IV delivery. In addition, siRNA and Au-Fe3O4 were released from the hydrogels and then delivered simultaneously to tumor sites in the form of NCs, which will aid a long-term gene/cyclic photothermal combination therapy. The effects of targeting moieties and hyperthermia on long-term gene silencing were also examined by western blot (Figure 5F). The suppression of PLK-1 protein


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indicated that the activity of siPLK-1 remained after long-term systemic delivery. More targeting moieties were involved, and more effective suppression of PLK-1 protein was obtained. The lowest expression was observed when hyperthermia was applied, indicating that the thermal energy enhanced silencing effect of siPLK-1. Previous studies show that cell sensitivity to hyperthermia is promoted through PLK-1 inhibition induced inactivation of heat shock transcription factor 1.36

Figure 5. Evaluation of in vivo tumor targeting and silencing effects of NCs released from the hydrogels. A) Real time fluorescence imaging of the tumor bearing mice and B) the corresponding quantitative analysis of cy5.5-tagged siPLK-1 target to the tumor tissues over time after SC injection of NC hydrogels or IV injection of NC solutions. C) Accumulation of the hydrogel released siRNA and Au-Fe3O4 nanoparticles within tumor tissues overtime. Tumor targeting efficiencies are calculated against injected dosage of siPLK-1 and Au-Fe3O4, respectively. D) Real time fluorescence imaging of the excised organs and E) the corresponding quantitative analysis of cy5.5 at 18 days post-SC injection of NC hydrogels or IV



injection of solutions. F) Immunoblot analysis of PLK-1 protein expression of tumor after long-term gene silencing by using NC hydrogels with different functional targeting. The symbol * indicates statistical significance levels of P< 0.05 and ** indicates P< 0.01.

Considering the short retention and fast excretion of NCs in the bloodstream after IV injection, the fluorescence intensity within tumor was also monitored in the first 24 h post-IV injection of the NC solutions. Non-significant difference of fluorescence intensities in the tumor tissues indicted the multiple targeting effect through IV delivery was limited compared to SC delivery (Figure 6A and B). Strong fluorescence intensities were observed in the kidney tissues of both mice types, highlighting the non-specific targeting of an IV injection approach, with low tumor targeting efficiency and potential toxicity (Figure 6C). Quantitative analysis revealed that triple-targeting reduced the accumulation of siRNA in the main organs (Figure 6D).


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Figure 6. A) Real time fluorescence imaging of the tumor bearing mice and B) quantitative analysis of cy5.5 over time after IV injection of NCs solutions. C) Real time fluorescence imaging of the excised organs and D) quantitative analysis of cy5.5 at 24 h post-IV injection. The symbol * indicates statistical significance levels of P< 0.05 and ** indicates P< 0.01.

In vivo anti-cancer therapy. To analyze the influence of multiple hyperthermic cycles under NIR laser irradiation, tumor temperature was measured during two cycles of treatments. To this end, two cycles of NIR laser irradiation were performed at day 7 and day 14 post-SC injection of the hydrogels, respectively, as enough NCs had been disseminated from the injection site to the tumor target by these time-points. The tumor temperature increased to a hyperthermic range (44 ± 1 ºC) within 5 min, which was maintained for 30 min during the first NIR laser treatment (Figure 7A). Faster rise and higher therapeutic range in the tumor temperature were measured during the second cycle, which can be attributed to the continuous supply of Au-Fe3O4 from the hydrogels to the tumor. In contrast, two cycles of NIR were performed one and three days after IV injection of the NC solutions. The tumor temperature heated to only 42 ºC, due to poor targeting efficiency, possibly hindering the therapeutic effects (Figure 7B). To investigate in vivo anti-cancer effects, tumor bearing mice were divided into six groups (n = 4) and subjected to different treatments (Figure 7C, Figure S9). Compared to continual tumor growth observed in the untreated mice and only two



cycles of NIR laser treated mice, the tumor volume was suppressed by NCs that were released from the hydrogels through dual targeting, and was further reduced when triple-targeting was applied. Moreover, the combination with two cycles of hyperthermia resulted in the strongest tumor inhibition, indicating the potential synergistic anti-cancer actions of hyperthermia and siPLK-1. In contrast, the combination therapy of NCs through IV delivery resulted in poor tumor inhibition, related to the low tumor targeting. The body weights for all the mice were maintained in a healthy range during treatment (Figure 7D). Compared to the conditions in our previous work,31 we achieved a complete tumor elimination in a low-dose siRNA by using two cycles of photothermal treatment and triple-tumor-targeting strategy. The therapeutic effects were further confirmed by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and Hematoxylin & Eosin (H&E) staining, respectively (Figure 7E). The apoptotic tumor cells, stained as green, were present at much higher levels in the group treated by the combination therapy using the hydrogels. The H&E staining also validated the tumor inhibition results by presenting large amounts of dead cells with condensed or fragmented nuclei. Meanwhile, the liver and kidney tissues did not show pathological abnormalities, suggesting that systemic delivery of siPLK-1 is safe (Figure S10).


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Figure 7. In vivo anti-cancer therapy. A) Tumor temperature elevation induced by two cycles of NIR laser irradiation at day 7 and day 14 post-SC injection of NC hydrogels, or B) at one and three days post-IV injection of NC solutions. C) Therapeutic outcomes of NC hydrogels or solutions on tumor xenografted mice. The tumor volume (V/Vinital) is plotted versus time after treatments. The red and blue arrows indicate the time points for two cycles of NIR laser after IV and SC injection, respectively. The symbol * indicates statistical significance levels of P< 0.05 and **



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indicates P< 0.01. D) The body weight records of mice. E) TUNEL and H&E stained tumor section excised 18 days after different treatments, respectively. The scale bars represent 100 μm.

■ CONCLUSIONS It is necessary to develop a new systemic drug delivery method because of the low tumor targeting efficiency through conventional intravenous (IV) delivery. We proposed biodegradable and thermosensitive nanocapsule (NC) hydrogels as an alternative

to

IV

triple-tumor-targeting,

delivery,

which

hydrogel-modulated

allow

for

sustained

EPR/folate/magnetism

release,

and

long-term

gene/cyclic photothermal combination therapy. The hydrogels were administrated through subcutaneous injection and worked as a ‘drug-release depot’ for the sustained release of NCs that can specifically target tumor. Compared to IV injection, we dramatically improved the tumor targeting efficiency more than 10% and reduced non-specific accumulation in healthy organs. The tumors were completely eliminated in vivo through the combination of our hydrogel system and two cycles of hyperthermia, compared to either single therapy or combination therapy using IV delivery of NC solutions. Our approach will improve the efficacy and safety of cancer therapy; i.e., long-term and effective tumor targeting while minimizing toxicity to the patient. We hope that other scientists are inspired to build upon our systemic drug delivery approach.


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■ MATERIALS AND METHODS Materials. Hexachlorocyclotriphosphazene (Sigma-Aldrich, USA) was purified by sublimation at 55 °C under vacuum. α-amino-ω-methoxy-poly(ethylene glycol) (AMPEG, MW=750 Da) was prepared as described previously.37 L-isoleucine ethyl ester hydrochloride (IleOEt·HCl) was purchased from A&Z Food Additives LTD (Hangzhou China). Aminoethanol (AEtOH), dimethylaminopyridine, succinic anhydride, folic acid, PEI (molecular weight = 800 Da, branch type), iron(III) acetylacetonate (97.0%), gold(III) chloride trihydrate, 1,2-dodecanediol (90%), oleic acid

(99%),

oleylamine

(70%),

dicyclohexylcarbodiimide

(DCC),

n-hydroxysuccinimide (NHS), and anhydrous N,N-Dimethylformamide (DMF, 99.8%) were all purchased from Sigma-Aldrich without further purification. Lipofectamine 2000 was acquired from Invitrogen. 1-octadecene (90%) was obtained from Alfa Aesar. Tetrahydrofuran (THF) and triethylamine (TEA) were purified under a dry nitrogen atmosphere by distilling and refluxing over sodium metal/benzophenone and barium oxide, respectively. All animal experiments were approved by the Animal Care Ethnic Committee of Korea Institute of Science and Technology. Cy5.5-tagged siRNA against PLK-1 was obtained from ST Pharm Co., LTD (Daejeon, Korea). PLK1 sense strand is 5’-GAUCACCCUCCUUAAAUAU-3’ and PLK1 anti-sense strand is 5’-AUAUUUAAGGAGGGUGAUC-3’. Synthesis of FPP. FPP polymer was synthesized based on the previous publication.31 In brief, poly(dichlorophosphazene) was dissolved in THF, to which IleOEt·HCl, TEA, AMPEG, and AEtOH were added stepwise followed by



acidification using succinic anhydride and conjugation with PEI. The carboxyl group of folic acid was activated by DCC/NHS in the anhydrous DMF before adding to the PEI conjugated poly(organophosphazene). After the reaction, the mixture was filtered and purified using a dialysis membrane (Spectra/Pro, MWCO: 10-12 kDa) against methanol and water for three days at 4 °C, respectively. The final FPP polymer was preserved at -20 °C after freeze drying. Synthesis and characterization of Au-Fe3O4 nanoparticles. Au-Fe3O4 nanoparticles were synthesized based on thermal decomposition32. In brief, 2 mmol Fe(acac)3, 10 mmol 1,2-dodecanediol, 6 mmol oleic acid, 6 mmol oleylamine, and 10 mL benzyl ether were mixed and stirred in a three-neck round-bottom flask connected to a flow of nitrogen at 120 ºC for 20 min. The deaerated gold precursor solution (0.1 mmol HAuCl4 + 1.5 mmol oleylamine + 5 mL benzyl ether) was injected to the flask when it was heated to 180 ºC and then maintained for 30 min. The color of solution turned to dark purple that indicated the formation of gold nanoparticles. At last, the mixture was heated to 298 ºC for 1 h. After the reaction, the mixture was cooled down to room temperature, precipitated and separated using ethanol/chloroform and centrifugation. The as-synthesized Au-Fe3O4 nanoparticles were dispersed in chloroform containing 20 μL oleic acid and oleylamine. The basic properties of Au-Fe3O4 nanoparticles were measured by TEM, scanning TEM, EDX, and vibrating sample magnetometer. Preparation of NC hydrogels. NC hydrogels were prepared by simple physical mixing of FPP polymer, siRNA, and Au-Fe3O4 nanoparticles. As-synthesized


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hydrophobic Au-Fe3O4 nanoparticles and FPP polymer were dissolved together in chloroform to ensure complete dissolution. Upon the complete evaporation of solvent under vacuum for 24 h, the mixed Au-Fe3O4 and FPP polymer were then dispersed in diethyl pyrocarbonate (0.1%) treated water containing siRNA at 4 ºC under the gentle stirring to form homogenous NC solutions that could be transformed into the hydrogels quickly at body temperature. Characterization of NC solutions and hydrogels. Morphology and particle size of the NC solutions (0.5 wt% FPP) was measured by TEM imaging (Tecnai G2 F20, FEI, Hillsboro, Oregon, USA). The sample solutions were loaded on a carbon-coated copper grid and negatively stained with 2% uranyl acetate for 1 min. The hydrodynamic average diameter and zeta potential of NC composed of different N/P ratio were then measured by DLS using a zetasizer (Malvern Instruments Ltd., Malvern, Worcestershire, UK). The N/P value is the ratio of moles of the amine groups of cationic polymers to phosphate groups of RNA. The UV absorption of FPP/Au-Fe3O4 NC solutions was measured by using a spectrophotometer (Model 8453, Agilent, USA). The sol-gel phase transition behavior of the hydrogels was evaluated by measurement of viscosity as a function of temperature using a viscometer (RVDV-III+, Brookfield, USA) in a shear rate of 0.1 s-1. Temperature elevation of Au-Fe3O4 nanoparticles under a NIR laser radiation was estimated. The FPP/N solutions (0.1 mL) were loaded into a 1.5 mL microtube covered by styrofoam to prevent heat dispersion. The samples were then exposed to the NIR laser at 808 nm



(Oclaro Inc., San Jose, CA, USA). The real-time temperature was recorded by using a digital 33-gauge needle thermocouple (Omega, Stamford, CT, USA). Cytotoxicity evaluation of NCs. MDA-MB-231 cancer cells were seeded at a density of 1 × 104 cells per well in a 96-well plate. After 24 h incubation at 37 ºC, complete RPMI 1640 medium was replaced by the medium containing NC solutions at varying concentrations up to 125 μg Fe/mL, 500 ng siRNA/mL, and 5 mg FPP/mL. Cells were cultured for 24 h at 37ºC with an externally applied magnetic field by placing a magnet (neodymium, L × W × H (cm) = 15 × 10 × 2.5) beneath the cell culture plate. Hyperthermia treatment was simulated by heating the cells in a water bath at 43 ºC for 20 min each time. Cell viability was then examined using MTT assay. The live/dead assay (L3224, Thermofisher Scientific, USA) was used to stain the cells and confirm the cytotoxicity induced by multiple hyperthermia under NIR laser irradiation at 43 ºC for 20 min each time. The pathway of cellular death by siPLK-1 was also examined by apoptosis/necrosis detection kit (ENZ-51002, Enzo Life Science, USA). The stained cells were observed using a confocal laser microscope (LSM 700, Carl Zeiss, Germany). Cellular uptake and gene silencing effect of NCs. MDA-MB-231 cancer cells (2 × 104 cells/well) were cultured in a 24-well plate for 24 h at 37 oC. The cells were treated by the RPMI 1640 medium containing NC or lipofectamine 2000 with 2.5 μg cy5.5-tagged siPLK-1 and a magnetic attraction. After 4 h culture, each well was replaced by fresh medium for another 44 h culture. The intensities of cy5.5 in the cells were observed by a confocal laser microscope. The gene silencing effect of siPLK-1


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was examined by using Western Blot. The protein concentration of cell lysates was measured by bicinchoninic acid (BCA) assay. Each sample was separated by electrophoresis on 10% SDS-polyacrylamide gel and transferred to a PVDF membrane (Millipore, Billerica, MA). The membrane was blocked with TBS containing 5% skim milk, followed by incubation with primary PLK-1 antibody overnight (Santa Cruz Biotechnology, CA, USA). After washing, the membrane was incubated with horseradish peroxidase-conjugated secondary antibody and the signals were detected with the ECL Western blotting detection reagents (GE Healthcare, Buckinghamshire, UK). The expression of PLK-1 was normalized with β-actin, and the band intensities were quantified by Image J software. In vitro release of NCs from hydrogels. NC hydrogels (100 μL, 6.5 wt% FPP, 500 μg Au-Fe3O4 nanoparticles) were loaded in a millicell with a pore size of 12 μm (Millipore, USA) and then immersed to 10 mL PBS (pH=7.4) under a gentle shake (50 rpm) in a water bath at 37 ºC. NCs released from the hydrogels should penetrate the pores of the millcell and disperse to the PBS environment. Morphology and particle size of the released NCs were examined by TEM and DLS, respectively. In vivo release and tumor targeting of NCs. The xenograft tumor model was established using 5-week old female BALB/c nude mice (NARA Animal Inc., Korea). The cell suspension (200 μL, 1:1 mix with Matrigel) (BD Bioscience, USA) containing 1 × 107 MDA-MB-231 cells were subcutaneously injected to left dorsal flank of the mouse. NC hydrogels (80 μL, 6.5 wt% FPP, 500 μg Au-Fe3O4, 100 μg cy5.5-tagged siPLK-1) were subcutaneously injected to right dorsal flank of mice



when the tumor volume reached 100 to 150 mm3. The effect of magnetic targeting was estimated by placing a disc magnet (neodymium, 9 mm dia. × 3 mm thick, Gauss: 12100) over the face of a tumor with a medical tape. The mice were sacrificed at the desired time and then the hydrogels were excised from the skin and weighed. The residual Au-Fe3O4 nanoparicles and siPLK-1 that remained in the hydrogels were measured using the potassium thiocyanide colorimetric analysis and the RiboGreen assay, respectively38-39. The Au-Fe3O4 nanoparicles targeted to the tumor was also accessed, simultaneously. The excised tumor tissues were freeze-dried, weighted, dissolved in nitric acid, mineralized, and diluted in distilled water. Iron concentrations of each sample were determined by AAS (ICE 3500, ThermoFisher Scientific, Waltham, MA, USA). To examine the tumor targeting of siRNA, the intensity of cy5.5-tagged siPLK-1 was monitored and quantified. Real-time fluorescence imaging of cy5.5-siPLK-1 that remained in the hydrogels and targeted tumor were both measured by IVIS spectrum (Caliper Life Sciences Inc., Hopkinton, MA, USA). The mice were sacrificed at 18 days post-SC injection or one day post-IV injection. The intensity of cy5.5 in the excised tumor and main organ tissues were measured and quantified by IVIS spectrum. In addition, the excised tumor tissues were then homogenized and centrifuged to obtain supernatant for the analysis of long-term gene silencing effect. The extracted proteins were quantified by BCA assay followed by Western Blot analysis. PLK-1 expression levels were normalized to β-actin, and the band intensities were quantified using Image J software.


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In Vivo anti-cancer effect. The tumor-bearing mice were divided randomly into six groups (n=4) as follows when the tumor volume reached 100 to 150 mm3: a = untreated, b = two cycles of NIR by SC injection of FPP/N hydrogels without siRNA, c = dual-targeting by SC injection of NC hydrogels, d = triple-targeting by SC injection of NC hydrogels, and e = triple-targeting by SC injection of NC hydrogels and two cycles of NIR, and f = triple-targeting by IV injection of NC solutions and two cycles of NIR. The injection volume of hydrogels (6.5 wt% FPP) and solutions (2.6 wt% FPP) were 80μL and 200 μL per mouse, respectively. The same concentrations of siPLK-1 (100 μg per mouse) and Au-Fe3O4 (500 μg per mouse) were applied in both SC and IV administration. The tumor temperatures were monitored by a digital needle thermocouple over the course of cyclic photothermal therapy. The first and second cycle of NIR were performed at day 7 and day 14 post-SC injection, or one and three days post-IV injection, respectively. The tumor volume was measured by a digital caliper manually, and calculated using the formula: Tumor volume (mm3) = 6/π × length × width × height. For histological analysis, the harvested tumor and main organ tissues were subjected to fix, dehydration, and paraffin section. The sectioned slices were stained by TUNEL and H&E, respectively. The iron accumulated in the tumor tissues was confirmed by Prussian Blue staining and counterstaining with Nuclear Fast Red.

■ ASSOCIATED CONTENT Supporting Information



The Supporting Information is available free of charge on the ACS Publications website at DOI: Characterization of Au-Fe3O4 nanoparticles, Structure of FPP polymer, UV-visible spectra of Au-Fe3O4, Au and Fe3O4 solutions, Response of FPP/N aqueous solutions to a magnetic force-assistant attraction, Temperature dependent viscosity variations of the hydrogels, Scanning TEM image of the released NCs from the hydrogels, In vivo dissolution of NC hydrogels after SC injection to the nude mice, Prussian blue staining for excised tumor tissues, Representative photographs of tumor-bearing nude mice before and after treatment, H&E and TUNEL staining of liver and kidney tissues ■ AUTHOR INFORMATION Corresponding Author *E-mail address: [email protected] ORCID Z. Q. Zhang: 0000-0002-5042-8342 Y. M. Kim: 0000-0002-0912-3010 S. C. Song: 0000-0001-9344-0686 Author Contributions Z. Q. Zhang and Y. M. Kim contributed equally to this work. Funding


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This research was financially supported by Korea Institute of Science and Technology (2E27930) and National Research Foundation of Korea (2014M3A9B16034220; 2017M3A9F5027614; 2018M3A9H1024872). Notes The authors declare no competing financial interests. ■ ACKNOWLEDGMENTS We greatly thank Dr. Yeon Kyung Lee to offer the NIR laser instruments. ■ REFERENCES (1) Ball, R. L.; Hajj, K. A.; Vizelman, J.; Bajaj, P.; Whitehead, K. A. Lipid Nanoparticle Formulations for Enhanced Co-delivery of siRNA and mRNA.

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Abstract Graphic


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Injectable and quadruple-functional hydrogel as an alternative

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