Research Papers

Influence of Hyperthermia on Efficacy and Uptake of Carbon Nanohorn-Cisplatin Conjugates

[+] Author and Article Information
Matthew R. DeWitt, Allison M. Pekkanen, John Robertson, Christopher G. Rylander

School of Biomedical Engineering and Sciences,
Virginia Tech–Wake Forest,
Blacksburg, VA 24061

Marissa Nichole Rylander

School of Biomedical Engineering and Sciences,
Virginia Tech–Wake Forest,
Blacksburg, VA 24061
e-mail: mnr@vt.edu

1Corresponding author.

Contributed by the Bioengineering Division of ASME for publication in the Journal of Biomechanical Engineering. Manuscript received October 7, 2013; final manuscript received December 17, 2013; accepted manuscript posted December 23, 2013; published online February 5, 2014. Editor: Victor H. Barocas.

J Biomech Eng 136(2), 021003 (Feb 05, 2014) (10 pages) Paper No: BIO-13-1471; doi: 10.1115/1.4026318 History: Received October 07, 2013; Revised December 17, 2013; Accepted December 23, 2013

Single-walled carbon nanohorns (SWNHs) have significant potential for use in photothermal therapies due to their capability to absorb near infrared light and deposit heat. Additionally, their extensive relative surface area and volume makes them ideal drug delivery vehicles. Novel multimodal treatments are envisioned in which laser excitation can be utilized in combination with chemotherapeutic-SWNH conjugates to thermally enhance the therapeutic efficacy of the transported drug. Although mild hyperthermia (41–43 °C) has been shown to increase cellular uptake of drugs such as cisplatin (CDDP) leading to thermal enhancement, studies on the effects of hyperthermia on cisplatin loaded nanoparticles are currently limited. After using a carbodiimide chemical reaction to attach CDDP to the exterior surface of SWNHs and nitric acid to incorporate CDDP in the interior volume, we determined the effects of mild hyperthermia on the efficacy of the CDDP-SWNH conjugates. Rat bladder transitional carcinoma cells were exposed to free CDDP or one of two CDDP-SWNH conjugates in vitro at 37 °C and 42 °C with the half maximal inhibitory concentration (IC50) for each treatment. The in vitro results demonstrate that unlike free CDDP, CDDP-SWNH conjugates do not exhibit thermal enhancement at 42 °C. An increase in viability of 16% and 7% was measured when cells were exposed at 42 deg compared to 37 deg for the surface attached and volume loaded CDDP-SWNH conjugates, respectively. Flow cytometry and confocal microscopy showed a decreased uptake of CDDP-SWNH conjugates at 42 °C compared to 37 °C, revealing the importance of nanoparticle uptake on the CDDP-SWNH conjugate's efficacy, particularly when hyperthermia is used as an adjuvant, and demonstrates the effect of particle size on uptake during mild hyperthermia. The uptake and drug release studies elucidated the difference in viability seen in the drug efficacy studies at different temperatures. We speculate that the disparity in thermal enhancement efficacy observed for free drug compared to the drug SWNH conjugates is due to their intrinsic size differences and, therefore, their mode of cellular uptake: diffusion or endocytosis. These experiments indicate the importance of tuning properties of nanoparticle-drug conjugates to maximize cellular uptake to ensure thermal enhancement in nanoparticle mediated photothermal-chemotherapy treatments.

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Grahic Jump Location
Fig. 1

(a) Depiction of single-walled carbon nanohorns in their aggregated state of many single-walled cones forming ∼150 nm diameter dahlia shape. Two types of CDDP-SWNHs conjugates were developed to determine their potential use as photothermal enhancers of the drug. The first SWNH-CDDP is the incorporated CDDP (b) in which the SWNHs are opened and CDDP is loaded into the internal space of the nanoparticle. The second conjugate (c) utilizes covalent attachment of the CDDP to the exterior surface of the SWNH. In order to visualize uptake kinetics, Zn/S shell Cd/Se quantum dots were attached to the SWNHs creating the SWNH-QD (d).

Grahic Jump Location
Fig. 2

(a) TEM image showing dahlia shape of pristine SWNHs with an average diameter of 80 nm. (b) TEM image showing high contrast QDs (dark aggregations) on an aggregate of SWNHs attached to lacey carbon mesh. (c) TEM illustrating successful incorporation of CDDP (dark aggregations) in SWNHs and (d) TEM showing dark attachment of CDDP to SWNH.

Grahic Jump Location
Fig. 3

Proliferation (viability) data for AY-27 cells exposed to one of three (a–c) CDDP regimens and the effect of mild hyperthermia on the efficacy of the treatment. (a) Viability for free CDDP exposed to the cells for 1 h for three heating profiles explained in methods and compares to the projected additive of mild hypothermia and CDDP. (b) Similar data for CDDP@SWNHs and (c) shows the viability when the cells are exposed to CDDP_INC_SWNHs. (d) Plots the difference in viability between 42 °C and 37 °C exposure for the three types and highlights the changes in thermal enhancement.

Grahic Jump Location
Fig. 4

Uptake of SWNH-QDs by AY-27 cells under normal physiological conditions (37 °C) and hyperthermia(42 °C) determined by FACs cytometry. (a) Median fluorescent intensity of the cells from 0–60 min exposure of heat and SWNH-QD. (b) Plots the percent positive cells determined by gating the fluorescent signal from (c) a control sample. (d) and (e) are representative data from 37 °C and 42  °C for 1 h, respectively, showing a sharp decrease in both fluorescence intensity and percent gated positive.

Grahic Jump Location
Fig. 5

Images of AY-27 cells exposed to SWNHs for 1 h taken 24 h after exposure. Brightfield images(a) and (d) 24 h after exposure to 0.01 mg/mL SWNH at 37 °C and 42 °C, respectively, showing nanoparticle aggregation within the cell after uptake(b) and (e) are stained for the cytoskeleton, showing internalization of the nanoparticles(red dots) at 37 °C and 42 °C, respectively, and (c) and (f) are DAPI stained to show internalization of the nanoparticles(red dots) reaches the nucleus after 24 h.

Grahic Jump Location
Fig. 6

Median fluorescent intensity for two different preheat stresses of 42  °C are compared to the control nonheated samples. Cells were allowed to rest for 1 h then were exposed to 0.01 mg/mL SWNH-QD for 1 h and fluorescent intensity was measured via FACs.

Grahic Jump Location
Fig. 7

Release of CDDP from CDDP_INC_SWNH (red and blue lines) and CDDP@SWNH(red dots along x-axis). The quantities of CDDP released into 400 mL water were measured via ICP-MS for platinum concentration. Results showed sufficient release of incorporated CDDP within 12 h, while no release of CDDP from the CDDP@SWNH was measured, showing stability of the bond.



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