A Particle-in-Particle Platform for Nanoconfinement-induced

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A Particle-in-Particle Platform for Nanoconfinement-induced Oncothermia Eunsoo Yoo, Jong Hoon Choi, Ngoc Ha Hoang, Jung Seok Lee, Steve Vuong, Byul Hur, Patrick Han, Kyung Taek Oh, Tarek M Fahmy, and Dongin Kim ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.8b00490 • Publication Date (Web): 09 Nov 2018 Downloaded from http://pubs.acs.org on November 11, 2018

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ACS Applied Bio Materials

A Particle-In-Particle Platform for Nanoconfinement-Induced Oncothermia Eunsoo Yoo1, Jong Hoon Choi1, Ngoc Ha Hoang1,5, Jung Seok Lee2, Steve Vuong1, Byul Hur4, Patrick Han2, Kyung Taek Oh5, Tarek Fahmy2,3, and Dongin Kim1* 1

Department of Pharmaceutical Sciences, Irma Lerma Rangel College of Pharmacy, Texas A&M University System Health Science Center, College Station, TX 77843, USA

2

Department of Biomedical Engineering and 3Immunobiology, Yale University, New Haven, CT 06511 USA 4

Department of Engineering Technology and Industrial Distribution (ETID), Texas A&M University, College Station, TX 77843 USA 5

College of Pharmacy, Chung-Ang University, Seoul, South Korea

KEYWORDS: Nanoconfinement, Particle-in-Particle, Oncothermia, Gold nanoparticle, Cancer

ACS Paragon Plus Environment

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ABSTRACT

Oncothermia, a special form of hyperthermia for oncological purposes, has been widely shown to be an effective mode of cancer therapy. Yet, its adoption amongst standard therapeutic practices has been limited by constraints in delivering sufficient thermal energy to tumor targets. To overcome these unique challenges in delivery presented by oncothermic therapeutics, we engineered a novel universal platform for hyperthermia cancer therapy utilizing versatile biocompatible materials. Herein, we show that Gold Particle-in-Particle (PIP), in which gold nanoparticles are physically confined within PLGA-PEG nanoparticles, significantly enhances thermal energy production by red-shifting the gold nanoparticle’s absorption spectra via a mechanism in which we call Nanoconfinement-Induced Therapeutic Enhancement (NITE). NITE mediated Gold PIPs significantly suppress breast, skin, and multi-drug resistant tumors and result in a multi-fold increase of heat shock protein expressed by cancer cells in vivo. Co-treatment of Gold PIP with doxorubicin shows a synergistic advantage. Using tumor-bearing mice, significant suppression of tumor growth by Gold PIPs shows the advantage of NITE mediated hyperthermia. Thus, we conclude that NITE mediated Gold PIP can be a strong anticancer therapy because of its sufficient amount of heat generation.

INTRODUCTION Despite various therapeutic and diagnostic developments, cancer remains a major public health problem worldwide and is the second leading cause of death in the United States. 1 Fortunately, novel therapeutic assessments continue to be developed and explored for the improvement of cancer patients’ mortality rates. However, a major obstacle in the widespread translation of many


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laboratory techniques to the bedside has stemmed from challenges in reconciling sufficient therapeutic efficacy of treatments with their associated systemic toxicity. 2-3 To achieve this precarious balance between efficacy and toxicity, new therapeutic modalities have been developed and tested and among them4-6, gold nanoparticles have been presented as a promising therapeutic agent for inducible hyperthermia7-8 as they are able to physically ablate their surrounding tissues when exposed to predetermined laser light, radio, or magnetic cues. Gold nanomaterials of various shapes and sizes such as spherical nanoparticles (AuNP) 9-10, nanorods (AuNR)8, 11, nanocages12-13, and nanoshells (AuNS)3, 7 have previously been shown to demonstrate multi-functional therapeutic nanoparticles due to their size controllability14-15, biocompatibility16, surface modularity17, and unique photothermal properties.13 Previously demonstrated oncothermia applications include antibody-conjugated AuNP for the treatment of benign and malignant epithelial cells18, polyethylene glycol (PEG) coated AuNS and anti-HER2 coated AuNS for targeting human breast carcinoma19 and AuNR decorated with folate for induction of tumor apoptosis in KB cells and NIH/3T3 cells death. 20 Furthermore, gold nanoparticles encapsulated PEG-PCL micelles were tested as a CT contrast agent and a sensitizer for radiation therapy. 21-22 Despite their broad versatility

and potential as a strong candidate for hyperthermia

therapy/oncothermia11, 23, gold nanoparticles have yet to be translated for clinical applications because of the following limitations: insufficient heat production, attenuated near-infrared (NIR) sensitivity in deep tissue, and difficulty of suppressing against both sensitive and multidrugresistant (MDR) tumors. Especially since individual gold spherical nanoparticles have absorbance wavelengths at 500~530 nm24 and it is way below the NIR (>800 nm) wavelengths for deep penetration of laser, they cannot be feasibly utilized for the in vivo photothermal treatments.25 Although increasing the size of spherical gold nanoparticles does indeed red-shift their absorbance,


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increasing particle size to 100 nm only moves the maximum absorption from 500 nm to 550 nm which would not be practical for clinical therapy. 24 The disadvantages of using gold nanorods11, nanocages12-13, and nanoshells7, 19 include loss of their original shape upon heating26, causing diminishing absorbance at NIR wavelength and thus reduced energetic output for inducible hyperthermia. Since current hyperthermia regimens only deliver individual gold nanoparticles to the targeted area, the total heat produced is insufficient for suppression of diseases. To overcome these physical and physiological barriers, a significant amount of gold nanoparticles must be administered.27 However, chances of systemic side effects increase with a dose which is enough to prevent translation into clinical applications. 28 Gold surface modifications via direct attachment of targeting ligands to bare gold surface present another challenge because it can cause photocarbonization of signal molecules of ligands or nearby impurity molecules/attached surface molecules under prolonged exposure to laser irradiation. 29 The last limitation of gold nanoparticles is their lack of stability in aqueous solvents. 30 Thus, it is of substantial importance to develop a novel platform which can overcome these hurdles in physics, thermodynamics, biocompatibility, and drug delivery associated with utilizing gold nanoparticles for inducible hyperthermia. Herein, we report a gold-based system that yields significant enhancements in hyperthermic temperature elevation through “Nanoconfinement-induced Therapeutic Enhancement” (NITE), a phenomenon uniquely arising from the encapsulation of multiple gold nanoparticles within another nanoparticle called Particle-in-Particle (Gold PIP). The PIP design leads to significant red-shifting of peak absorption wavelengths as well as enhanced heat production, thereby allowing deeper penetration of laser within tumor tissues and sufficiently curbing normal disease progression. 31 A biocompatible polymeric shell of PIP, such as poly(lactic-co-glycolic acid) (PLGA)-PEG micelle32 coating around the gold nanoparticles, is shown to maintain particle stability while also providing


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a conjugatable surface for attachment of ligands for enhanced targeting specificity. Spherical gold nanoparticles encapsulated within Gold PIP also provide additional support to its structural integrity during heating, maintaining its capacity for inducible hyperthermia.

RESULTS Preparation and Characterization of Gold PIP For the formulation of Gold PIP, gold clusters were developed by the aggregation of gold nanoparticles after addition of PBS and subsequent freeze-drying process (Figure 1a). To select the appropriate carrier for encapsulating gold nanoclusters, we formulated gold nanoclusters in the presence of Pluronics with various hydrophilicity33 or PLGA-PEG micelles.32 All of the samples with Pluronics yielded irregular shapes of gold nanoclusters. Conversely, PLGA-PEG formed spherical micelles that neatly encapsulated gold nanoclusters (Figure S1). Thus, PLGA-PEG was selected for subsequent formulations of Gold PIP for the study. Isolated gold nanoparticles (IGN) with 5 nm size showed a reddish color while gold nanoclusters (GNC) and Gold PIP showed a dark blue color. The transmission electron microscopy (TEM) images illustrated that 5 nm IGN were a spherical shape with ~5 nm diameters and Gold PIP has also a spherical shape but the gold nanoclusters without PLGA-PEG showed an irregular aggregation (Figure 1b). The dynamic light scattering (DLS) measurements confirmed that the average diameter of Gold PIP with 5 nm IGN (out of working range which is greater than 10 nm diameters) is shown ~150 nm with a narrow polydispersity index (PDI) while the GNC showed a multi-modal distribution (Figure 1b and Table 1). The zeta potential of Gold cluster and Gold PIP in PBS were (-) 41.3 and (-) 20.7 mV, respectively. It is indicating that the charge shielding due to the PLGA-PEG micelle (Table 1).34-35 Absorbance spectra of each formulation (Gold PIP and


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IGN) were scanned to determine their optical shift (Figure 1c). IGN showed a maximum wavelength at about 550 nm with a minimum absorbance at near-infrared (NIR) wavelengths.24 Interestingly, Gold PIP exhibited an absorbance shift to a NIR region (~800 nm) 36 which was similar to the gold nanorod25 and thereby allowing Gold PIP to be utilized for applications in deep tissues. Also, Gold PIP also showed a near ten-fold enhancement of NIR absorbance to IGN at equivalent gold concentrations (Figure 1c).

Nanoconfinement-mediated Heat Production Using the absorbance wavelength of Gold PIP, we determined the heat production of Gold PIP in response to NIR laser (~803 nm) measured by an infrared (IR) thermal camera and temperature probe. In this study, temperature increment or temperature profile of Gold PIP was compared to those of IGN and the water according to the laser irradiation time, the distance between laser and sample, and laser strength (Figure 1d ~ 1h and S2). Upon five minutes of NIR application, the temperature of Gold PIP reached up to ~60 °C which was significantly above the effective hyperthermia temperature range (41~45 °C) while IGN reached below 45 °C (Figure 1d).37-38 Thermal images (Figure 1d) also confirmed the highest temperature increments observed with Gold PIP. Since the highest temperature (above 60 °C) of Gold PIP was achieved when the laser and sample were 2 cm apart (Figure 1e), we maintained this distance for the rest of our experiments. The temperature increment or profile of Gold PIP was directly dependent on sample concentration and laser power (Figure 1f, g, and h). From our laser power dependent temperature results, we observed that Gold PIP can produce significant thermal energy while IGN and water showed minimum energy at 0.8 W. To confirm the NIR laser’s effect on the release kinetics of doxorubicin (DOX) from Gold PIP for possible synergistic combination therapy, we also measured


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the DOX release kinetics from DOX-encapsulated Gold PIP (Figure 1i). The DOX loading capacity was 450 g/mL, and its encapsulation efficiency against Gold PIP showed 5 wt % as DOX weight over micelle weight.


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a PLGA-PEG

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0.7 1.4 2.1 Power (W)

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Figure 1. Gold PIP Formulation and Its Optical and Thermal Advantage (a) Gold nanoclusters (GNCs) was prepared from isolated gold nanoparticles (IGN). GNCs were encapsulated into PLGA-PEG micelle to make Gold Particle-in-Particle (PIP). (b) The particle size and morphology of IGN, GNC, and Gold PIP were determined by dynamic light scattering and TEM, respectively. (c) The absorbance IGN, Gold PIP, and Gold Nanorod with the same gold concentration was scanned from 400 to 1000 nm. (d) The temperature enhancement of water, IGN, and Gold PIP were determined by a thermal camera. (e) The distance between sample and laser dependent heat production was validated for five minutes of laser irradiation. (f) The laser power dependent heat production of water, IGN, and Gold PIP was measured for five minutes of laser irradiation. (g) Heat production kinetics of Gold PIP was investigated according to its concentration. (h) Laser power dependent heat production kinetics of Gold PIP was determined. (i) Doxorubicin (DOX) release kinetics was determined by using Gold PIP and PLGA-PEG micelle without gold that encapsulated DOX, respectively. At six hours of incubation, laser irradiation was applied for five minutes to all samples. The DOX encapsulation amount was 450 g/mL, and its encapsulation efficiency against Gold PIP was 5 wt % (DOX weight/micelle weight).

The observed results indicated that a burst release of drug was observed when the NIR laser was applied, suggesting that heat from Gold PIP was triggering payload release. Therefore, these results conclude that NITE Gold PIP can boost thermal energy production profiles proving itself a strong candidate for hyperthermia applications. These improved characteristics likely originated from two key factors: 1) nanoconfinement mediated sensitivity to NIR range of the laser, and 2) nanoconfinement of the gold particle within PLGA-PEG micelle.


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Table 1. The particle sizes and zeta potential of gold nanoclusters and Gold PIP were determined by DLS.

Sample

Size (nm)*

PDI

Zeta (ζ) Potential

Gold nanocluster (GNC)

242.3 ± 3.9

0.387 ± 0.029

(-) 41.3 ± 1.058

Gold PIP

157.4 ± 1.0

0.128 ± 0.009

(-) 20.7 ± 1.078

* Determined by DLS, mean ± standard deviation

Mechanism of NITE Our results indicated that the nanoconfinement mediated an enhanced heat production exhibiting strong potentials for anticancer hyperthermia. Thus, we designed a series of experiments to illustrate the mechanisms of NITE in this study. First, Gold PIP was disrupted to make gold nanoparticles free in the solution and the absorbance, temperature, and particle size were determined. Upon disruption of Gold PIP, a 15 % loss of absorbance was observed compared to the original Gold PIP as well as a diminished temperature from 47 °C to 40 °C (Figure 2a and b). DLS analysis also indicated that particles of a smaller size range (50~80 nm) appeared after disruption of Gold PIP suggesting freed gold nanoclusters or gold nanoparticles (Figure 2c). We also set up an experiment to prove NITE mediated heat production by conjugating gold nanoparticles with different numbers (0, 2, 4, and 8) of PEG arms, simulating confinement atmosphere experienced by gold nanoparticles. PEG of higher branching number which confined more gold nanoparticles within a given space showed higher heat production (Figure 2d). Collectively, these results prove nanoconfinement mediated heat production enhancement in which the temperature production of gold is proportional to its packing density within another particle.


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Since gold nanorods have unique NIR (700~900 nm) sensitivity due to its shape, it has been considered one of the prime candidates for hyperthermia in deep tissue application. However, its significant disadvantage is from the damage of the original rod shape after multiple rounds of NIR laser stimulation causing major loss of absorbance at NIR region and no heat production and making it impractical for clinical hyperthermia therapy. We thus measured and compared heat production of Gold PIP and gold nanorod after multiple NIR applications (Figure 2e). During the second NIR application, temperature (~53 °C) of Gold PIP maintained as was during the first NIR application, while gold nanorod showed attenuated temperatures from 47 °C to 43 °C during 2nd NIR application. TEM images also demonstrate a structural loss of rod shape (ii) (Figure 2e). We also confirmed the loss of intensity at maximum absorbance peak in the NIR region of the gold nanorod after multiple rounds of NIR applications while Gold PIP preserves its original intensity (Figure 2e). Thus, Gold PIP had better thermal stability than gold nanorod, leading to favorable properties for hyperthermia treatment.


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Figure 2. Proof-of-concept Studies for Nanoconfinement advantage (a) The absorbance of Gold PIP was scanned before and after its disruption. (b) Heat production of Gold PIP was determined before and after its disruption to prove the nanoconfinement advantage. (c) The particle size analysis of Gold PIP before and after its disruption was measured and it showed around 50 ~ 80 nm smaller particle size range. (d) Multi-arms of PEGs were conjugated with gold nanoparticles to artificially mimic the nanoconfinement environment, and each sample was under the laser irradiation for five minutes to take the heat production images (**p

A Particle-in-Particle Platform for Nanoconfinement-induced

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