Multifunctional polymeric nanoparticles for combined chemotherapeutic and near-infrared photothermal cancer therapy in vitro and in vivo

Abstract

Multifunctional Taxol-loaded PLGA nanoparticles show chemotherapeutic and photothermal destruction of cancer cells in vitro and in vivo.

---

Due to their ability to simultaneously provide detection, diagnosis, and therapy in a single nanoparticle, multifunctional nanomaterials have been intensively explored for promising applications in biomedicine.¹ Recently, nanoparticles with photothermal therapeutic capabilities have attracted a great deal of attention in the photothermal treatment of tumor cells.² Those near-infrared (NIR) resonant nanomaterials are chosen due to the optimal transmission to tissue in the NIR region. However, there are still some challenges existing for clinical applications. For instance, the efficient area of the photothermal destruction of cancer cells is restricted due to the limited laser beam spot, resulting in a confined therapeutic area of cancer tissues. To overcome this limitation, chemotherapy by the photothermal-control drug release method could be incorporated with NIR therapy in thermoresponsive polymers.

The combination of chemotherapeutics and hyperthermia has been an emerging approach for cancer therapy.³ However, this combinatorial therapy is highly requisite to deliver drugs and localized heating to the cancerous area. Nanomaterials hold great potential to achieve localized treatments to the area of interest. Recently, we have developed stabilizer-free poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs), and they can be easily and directly made to conjugate on the surface and the inner core can encapsulate drugs to potentially serve as multifunctional probes.⁴ In this work, we demonstrate the first example of nanosystems with the combination of chemotherapeutic and photothermal therapy in mammalian cells and in vivo in an animal model.

The stabilizer-free Taxol (paclitaxel)-loaded PLGA NPs were conjugated with amine-terminal Fe₃O₄ NPs (∼6 nm)⁵ and quantum dots (QDs) (∼12 nm) to obtain QD/Fe₃O₄/Taxol-loaded PLGA NPs with optical and MR imaging functionalities. Subsequently, poly(styrenesulfonate) (PSS)-coated Au nanorods with an aspect ratio of 3.9 were introduced to attach to the QD/Fe₃O₄/Taxol-loaded PLGA NP surface. Because Au NRs can absorb NIR light and convert it to heat, spherical PLGA NPs can be destroyed to efficiently release encapsulated Taxol. Both in vitro and in vivo studies were conducted to evaluate therapeutic performance. It was found that the combination of photothermal destruction and chemotherapy in the treatment of cancer cells provided effective therapy as compared to photothermal destruction or chemotherapy alone.

The transmission electron microscopy (TEM) images of the derived PLGA NPs are shown in Fig. 1. Fig. 1a shows Taxol-loaded PLGA NPs of ∼83 nm in diameter, which is approximately the same size as bare PLGA NPs (see ESI Fig. S1a†). Amine-terminal Fe₃O₄ NPs (∼1.45 pmole per PLGA mg) were attached to the surface of PLGA NPs, leading to a particle size of ∼130 nm for Fe₃O₄/PLGA NPs, as shown in Fig. 1b. NH₂-PEG-QDs (see ESI Fig. S1b†) with ∼56 pmole per PLGA mg were added to form QD/Fe₃O₄/PLGA NPs (Fig. 1c). The QD/Fe₃O₄/PLGA NPs had an average size of ∼156 nm. It was found that the surface conjugation with Fe₃O₄ and QD NPs resulted in conformation expansion of soft PLGA templates. The negatively charged PSS-modified Au NRs (∼0.0037 pmole per PLGA mg) were then electrostatically adsorbed on the QD/Fe₃O₄/PLGA NPs, bearing –NH₃⁺ groups that originated from QDs and Fe₃O₄ nanoparticles. Fig. 1d shows Au NR/QD/Fe₃O₄/PLGA NPs with a size of ∼193 nm. Additional TEM images and hydrodynamic diameters measured by dynamic light scattering spectrometry of the derived PLGA NPs can be seen in ESI Fig. S2 and S3.† The derived PLGA NPs were suspended and stable in the range of pH 5–8 and in 0.01–0.5 M of NaCl aqueous solution. It is expected that Au NRs covered over onto QDs and Fe₃O₄ nanoparticles. The Au NR/QD/Fe₃O₄/PLGA NPs exhibited the characteristic surface plasmon bands (∼520 and ∼800 nm) and the fluorescent intensity of Au NR/QD/Fe₃O₄/PLGA NPs was quenched in some degree (∼30% intensity decrease) after being coated with Au NRs as compared to QD/Fe₃O₄/PLGA NPs (see ESI Fig. S4 and S5†). Temperature evaluation profiles as a function of the laser irradiation period show Au NR/QD/Fe₃O₄/PLGA NPs with effective temperature elevation, reaching 40 °C after 1.5 min. No temperature increase was observed in the other derived PLGA NPs in the absence of Au NRs (see ESI Fig. S6†).

Fig. 1 TEM images of (a) stabilizer-free Taxol-loaded PLGA NPs stained with 2% phosphotungstic acid, (b) Fe₃O₄/PLGA NPs, (c) QD/Fe₃O₄/PLGA NPs (Fe₃O₄: white arrow, QDs: black arrow), and (d) Au NR/QD/Fe₃O₄/PLGA NPs.

The spherical morphology of Au NR/QD/Fe₃O₄/PLGA NPs was destroyed due to the local heat produced by irradiation using a diode CW laser (30 W cm⁻²) at 808 nm for 7 min (see ESI Fig. S7†). The generated heat induced PLGA NPs to melt and degrade. This indicates that PLGA NPs can be employed as photothermal-control drug release carriers. To examine drug release upon NIR irradiation, the Taxol release of the Au NR/QD/Fe₃O₄/Taxol-loaded PLGA NPs was monitored as a function of laser irradiation period (see ESI Fig. S8†), where the release of entrapped Taxol occurred fast upon a short period of laser irradiation while no apparent release was observed without exposure to laser.

In this study, the drug Taxol was loaded into the PLGA NPs to measure chemotherapeutic performance. The Taxol-loaded PLGA NPs and Au NR/QD/Fe₃O₄/Taxol-loaded PLGA NPs were evaluated for their release behavior in the absence of photo-irradiation. The amount of Taxol in PLGA NPs (% w/w) was about 3.6%. The released amount of Taxol from Au NR/QD/Fe₃O₄/Taxol-loaded PLGA NPs was only half of that from Taxol-loaded PLGA NPs (see ESI Fig. S9†). The surfaces of Au NR/QD/Fe₃O₄/Taxol-loaded PLGA NPs were covered with Au NRs, QDs, and Fe₃O₄ nanoparticles, which hindered drug release as compared to Taxol-loaded PLGA NPs. We also investigated the toxicity of Au NR/QD/Fe₃O₄/PLGA using HeLa cells (cervix cancer cells) in an MTT assay (see ESI Fig. S10†). It was found that hybrid PLGA NPs treated with HeLa cells showed no toxicity after an incubation of 24 h in various PLGA concentrations.

To achieve selective cancer cell targeting, anti-Her2 monoclonal antibodies were immobilized on the surface of Au NR/QD/Fe₃O₄/Taxol-loaded PLGA NPs. Fig. 2 shows cancer cell viability after treatment with derived PLGA NPs with/without laser irradiation (30 W cm⁻²) at a laser wavelength of 808 nm for 7 min. Notably, the cell viability was measured relative to a control group where the HeLa cells without the treatment of Ab/Au NR/QD/Fe₃O₄/Taxol-loaded PLGA NPs were also incubated for 24 h. Ab/Au NR/QD/Fe₃O₄/Taxol-loaded PLGA NPs did not induce apparent cell death after 24 h of incubation in various PLGA concentrations in the absence of laser irradiation. Considering the proliferation of cancer cells observed for the control group after 24 h incubation, it seems that the process involved in the gradual release of Taxol was not effective in the inhibition of cancer cell growth. For Ab/Au NR/QD/Fe₃O₄/PLGA NPs without the encapsulation of Taxol under laser irradiation, the cell viability decreased as PLGA concentration increased. A limited number of damaged cells (∼88% viability at 11.6 μg μL⁻¹ of PLGA concentration) was observed. The limited photothermal destruction is possibly due to cell damage being mainly restricted to the laser illumination beam spot because of Au nanorods absorbing laser irradiation. When Ab/Au NR/QD/Fe₃O₄/Taxol-loaded PLGA NPs received laser exposure, the cell viability dropped to ∼74% at 11.6 μg μL⁻¹ of PLGA concentration. This indicates that the Taxol burst release after laser irradiation produced an additional chemotherapeutic effect. As seen in Fig. S7 and S8,† the irradiated PLGA NPs resulted in fragmentation in morphology, giving a large release of Taxol yielding anticancer activity. Interestingly, a significant improvement in the destruction of cancer cells was observed when the irradiated PLGA NPs were incubated with HeLa cells for an additional 24 h before the measurements of cell viability. The cell viability was found to be ∼10% at 11.6 μg μL⁻¹ of PLGA. It seems that post-irradiation incubation allowed the released Taxol to more effectively kill distributed HeLa cells. It should be noted that no damaged cells were observed under NIR irradiation for HeLa cells only in the control groups. The parallel experiments were conducted using the same derived PLGA nanoparticles in the absence of anti-Her2 antibody conjugation all leading to over 95% of cell viability.

Fig. 2 HeLa cell viability (MTT assay) after treatment with Ab/Au NR/QD/Fe₃O₄/PLGA NPs and Ab/Au NR/QD/Fe₃O₄/Taxol-loaded PLGA NPs in various PLGA concentrations.

The laser confocal images showed the results of Ab/Au NR/QD/Fe₃O₄/Taxol-loaded PLGA NPs incubation with HeLa cells. Ab/Au NR/QD/Fe₃O₄/Taxol-loaded PLGA NPs incubated with HeLa cells successfully targeted the cell surface (see ESI Fig. S11a†). When the PLGA NPs-targeted HeLa cells were exposed to laser irradiation, the HeLa cell structure was seriously fractured (see ESI Fig. S11b†). Apoptosis was observed from the nuclear fragmentation and cytoplasm damage.

With in vitro efficacy results, we used Au NR/QD/Fe₃O₄/PLGA and Au NR/QD/Fe₃O₄/Taxol-loaded PLGA NPs for preliminary in vivo studies. Since both hybrid PLGA NPs were intratumorally injected into transplanted tumors of mice at this stage, no anti-Her2 antibody was conjugated on the particles surface. Male mice bearing tumors one week after transplantation, a subcutaneous dorsal flank injection of tumor cells (MBT-2 bladder cancer cells), received with/without laser illumination. The variations in tumor size are shown in Fig. 3. For PBS injection followed by laser irradiation, the tumor sizes continually grew. Compared to PBS injection, Au NR/QD/Fe₃O₄/Taxol-loaded PLGA NPs (100 μg/Kg) injection without laser irradiation displayed some anticancer effect as days prolonged, showing the suppression of tumor growth although the tumors still grew. This suggests that chemotherapeutic Taxol slowly released from Au NR/QD/Fe₃O₄/Taxol-loaded PLGA NPs restrained a certain degree of tumor growth, particularly in the latter days of the test period. For the photothermal treatment only, Au NR/QD/Fe₃O₄/PLGA with Taxol free NPs injection followed by laser irradiation produced a greater anticancer effect as compared to chemotherapeutic treatment only. However, the tumors seemed to start to grow in the latter days of the test period. A significant enhancement of the anticancer effect was observed when chemotherapy and photothermal destruction were combined, with Au NR/QD/Fe₃O₄/Taxol-loaded PLGA NPs injection being followed by laser irradiation. The tumor growth was apparently suppressed and the tumors tended to shrink as the test period went on. The mice treated with chemotherapy and photothermal destruction remained alive after two months, where the tumors of those treated mice either decreased 100% or showed no sign of regrowth after therapy. Additionally, we found that the size of the laser beam spot, which is related to the amount of Au NR/QD/Fe₃O₄/Taxol-loaded PLGA NPs being irradiated, on the tumor strongly affects anticancer performance. When the laser beam spot was narrowed from 12 mm² to 1 mm², effective tumor shrinkage was observed only in the early days of the test period but the tumors grew significantly at test days prolonged. This might suggest that a larger laser beam size, irradiating more PLGA NPs, would result in more effective anticancer activity. More detailed in vivo studies need to be carried out. The tumor size in each condition was controlled in the range of 70–150 mm³ prior to particles intratumoral injection.

Fig. 3 Relative tumor volumes were monitored as a function of post-irradiation days. The results for five mice are shown for each condition and the error bars were derived from the data for five mice. The tumors were exposed to a laser with a wavelength of 808 nm using a laser power density of 3 W cm⁻² for 7 min. (Note: the x-axis of Au NR/QD/Fe₃O₄/Taxol-loaded PLGA NPs without laser irradiation group represents the days after intratumoral injection of Au NR/QD/Fe₃O₄/Taxol-loaded PLGA NPs).

Since the Fe₃O₄ NPs are decorated on PLGA NPs, Au NR/QD/Fe₃O₄/Taxol-loaded PLGA NPs can potentially serve as a contrast agent for MR imaging. The r₁ and r₂ relaxivities of Au NR/QD/Fe₃O₄/Taxol-loaded PLGA NPs were determined to be 0.19 and 14.05 s⁻¹ mM⁻¹, respectively, and the T₂-weighted imaging intensity substantially darkened with increasing iron ion concentrations using a 3T MR system (see ESI Figs. S12 and S13†). The Au NR/QD/Fe₃O₄/Taxol-loaded PLGA NPs were further administered to A549 (lung cancer cells)-induced SCID mice. The same administered dosage of 100 μg/Kg as those used in Fig. 3 was intratumorally injected into mice. Fig. 4 shows the T₂-weighted images of A549-induced SCID mice before and after injection of Au NR/QD/Fe₃O₄/Taxol-loaded PLGA NPs. At immediately post-injection, the tumor area appeared darkened and the imaging signal decreased about 24%.

Fig. 4 In vivo MR T₂-weighted images of A549 (lung cancer cells)-induced SCID mice administered by intratumoral injection of the Au NR/QD/Fe₃O₄/Taxol-loaded PLGA NPs at (a) pre-injection and (b) immediate post-injection. (The arrows indicate the induced tumor area).

Notes and references

1. (a) J. Gao, W. Zhang, P. Huang, B. Zhang, X. Zhang and B. J. Xu, J. Am. Chem. Soc., 2008, 130, 3710–3711; (b) M. K. Yu, Y. Y. Jeong, J. Park, S. Park, J. W. Kim, J. J. Min, K. Kim and S. Jon, Angew. Chem., Int. Ed., 2008, 47, 5362–5365; (c) J. H. Lee, B. Schneider, E. K. Jardon, W. Liu and J. A. Frank, Adv. Mater., 2008, 20, 2512–2516.
2. (a) A. P. Alivisatos, Nat. Biotechnol., 2004, 22, 47; (b) X. Huang, I. H. El-Sayed, W. Qian and M. A. El-Sayed, J. Am. Chem. Soc., 2006, 128, 2115–2120; (c) J. Chen, D. Wang, J. Xi, L. Au, A. Siekkinen, A. Warsen, Z. Y. Li, H. Zhang, Y. Xia and X. Li, Nano Lett., 2007, 7, 1318–1322.
3. (a) R. D. Issels, M. Schlemmer and L. H. Lindner, Curr. Oncol. Rep., 2006, 8, 305–309; (b) D. Coffey, R. Getzenberg and T. DeWeese, JAMA, J. Am. Med. Assoc., 2006, 296, 445–448.
4. F. Y. Cheng, S. P. H. Wang, C. H. Su, T. L. Tsai, P. C. Wu, D. B. Shieh, J. H. Chen, P. C. H. Hsieh and C. S. Yeh, Biomaterials, 2008, 29, 2104–2112.
5. D. B. Shieh, F. Y. Cheng, C. H. Su, C. S. Yeh, M. T. Wu, Y. N. Wu, C. Y. Tsai, C. L. Wu, D. H. Chen and C. H. Chou, Biomaterials, 2005, 26, 7183–7191.

---

Footnote

† Electronic supplementary information (ESI) available: Experimental details. See DOI: 10.1039/b919172k

---