Preparation and Size Control of Sub-100 nm Pure Nanodrugs

Dec 16, 2014 - Nano-organic Photoelectronic Laboratory, Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Beijing. 100190, Pe...
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Preparation and Size Control of Sub-100 nm Pure Nanodrugs Jinfeng Zhang,† Yanan Li,‡ Fei-Fei An,‡ Xiaohong Zhang,*,§ Xianfeng Chen,† and Chun-Sing Lee*,† †

Center of Super-Diamond and Advanced Films (COSDAF) & Department of Physics and Materials Science, City University of Hong Kong, Kowloon, Hong Kong SAR, People’s Republic of China ‡ Nano-organic Photoelectronic Laboratory, Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Beijing 100190, People’s Republic of China § Institute of Functional Nano & Soft Materials Laboratory (FUNSOM) and Jiangsu Key Laboratory for Carbon-Based Functional Materials & Devices, Soochow University, Suzhou 215006, People’s Republic of China S Supporting Information *

ABSTRACT: Pure nanodrugs (PNDs), nanoparticles consisting entirely of drug molecules, have been considered as promising candidates for next-generation nanodrugs. However, the traditional preparation method via reprecipitation faces critical challenges including low production rates, relatively large particle sizes, and batch-to-batch variations. Here, for the first time, we successfully developed a novel, versatile, and controllable strategy for preparing PNDs via an anodized aluminum oxide (AAO) template-assisted method. With this approach, we prepared PNDs of an anticancer drug (VM-26) with precisely controlled sizes reaching the sub-20 nm range. This template-assisted approach has much higher feasibility for mass production comparing to the conventional reprecipitation method and is beneficial for future clinical translation. The present method is further demonstrated to be easily applicable for a wide range of hydrophobic biomolecules without the need of custom molecular modifications and can be extended for preparing all-in-one nanostructures with different functional agents. KEYWORDS: Drug delivery, pure nanodrugs, template, mass production, size control

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a major hurdle for animal and clinical trials. Meanwhile, it is well known that the size of a nanomedicine critically affects its accumulation, penetration, and efficacy in tumors.26−31 Nanocarrier-based drugs with sizes below 20 nm have been demonstrated to possess much improved in vivo pharmacokinetics with superior penetration and retention behaviors, decreased reticuloendothelial system (RES) uptakes, and enhanced blood circulations.32−34 However, PNDs sized below 100 nm was not achieved until the recent breakthrough by Kasai et al.16 They modified the drug−solvent physical interactions by jointing two SN-38 drug molecules to form dimers and obtained 30−50 nm particles upon reprecipitation. Although this approach provides the first workable solution for obtaining sub-100 nm PNDs, extending this to other drug molecules would require individual custom-designed molecular modifications. Furthermore, sub-20 nm PNDs and precise size control are yet to be demonstrated. To realize the full potential of PNDs, new synthesis approaches are needed to address the deficiencies of the current reprecipitation method. Particularly, we need a more general approach to prepare various PNDs with (1) precisely controlled sizes and ideally covering also the sub-20 nm range

ancer, as one of the most devastating diseases, has over 10 million new cases every year worldwide.1 Although chemotherapy has been extensively used, current anticancer drugs typically kill both cancer and healthy cells. Thus, one pressing challenge is how to deliver enough drugs to cancer cells and minimize their damage to healthy ones. Due to the enhanced permeability and retention (EPR) effect of nanomaterials, a wide variety of therapeutics based on nanodrugs have been introduced for targeted drug delivery to cancer tissues.2−5 In particular, the use of different nanocarriers for effective transportation of hydrophobic pharmaceutical agents to tumor sites has garnered major attention.6−12 Despite the merits of these nanocarriers, further studies are needed for improving their drug loading capacities (typically less than 10%), reducing potential systemic toxicity and understanding of their metabolisms and secretions.13−15 To address these issues, one desirable strategy is not to use any “carriers” for drug delivery, and this leads to development of pure nanodrugs (PNDs), nanoparticles (NPs) consisting entirely of drug molecules.16−20 PNDs are most commonly prepared by reprecipitation in which solutions of pure drugs in solvents of high solubilities are added dropwise into solvents of low solubilities.21−23 Although this reprecipitation technique is very simple, it faces critical challenges including low production rates, relatively large particle sizes (typically ∼100 to 500 nm) and batch-to-batch variations.24,25 In fact, the low production rate of this method is © XXXX American Chemical Society

Received: September 18, 2014 Revised: December 15, 2014

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Figure 1. Schematic illustration of the procedures of preparing pure nanodrugs with the aid of AAO templates. SEM images of VM-26 nanodrugs with different sizes of 10−20, 30−40, 70−80, and 90−100 nm. Insets are corresponding size distribution and polydispersity index (PDI) data. Scale bar is 500 nm.

Figure 1 illustrates the procedures for fabricating PNDs via the AAO template-assisted strategy. AAO templates (purchased from Chao Wei Nano Technology, China) with different pore sizes were first thoroughly washed with organic solvents to remove dusts and impurities. The AAO templates were then immersed in drug solutions of certain concentrations for 10 min. The soaked templates were then removed from the drug solutions followed by solvent evaporation for 10 min. The soaking and evaporation steps were repeated 10−20 times. The samples were then immersed in a dilute NaOH aqueous solution (0.001 M) for dissolving the AAO templates. Subsequently, drug nanoparticles were collected via 5−10 cycles of centrifugation, supernatant removal, and redispersion or by dialysis. Finally, the extracted PNDs were dispersed in deionized water by sonication. Supporting Information Figure S1 shows SEM images of AAO templates before and after loading of VM-26. An energy dispersive X-ray (EDX) spectrum (Supporting Information Figure S2) evidence the existence of all expected elements (C, O, and S) in the as-prepared PNDs. To illustrate the size tunability of this method, we prepared VM-26 PNDs with different sizes. Figure 1 indicates that the asprepared PNDs possess a spherical shape and comparatively monodisperse distribution with average sizes ranging from 17, 38, and 76 to 97 nm with polydispersity index (PDI) values of 0.182, 0.176, 0.193, and 0.188 respectively. It is noted that we successfully fabricated PND sizes smaller than 20 nm for the first time (see also TEM images in Supporting Information Figure S3a). Such a small size is attributed to the corresponding template pore dimension that would restrict continuous growth of the nanoparticles. It was also observed that the size of PNDs

and (2) higher production rates to allow direct clinical trials without further tedious and time-consuming concentration procedures and cost reduction. Here, we demonstrate a simple anodized aluminum oxide (AAO) template-assisted process that satisfies these requirements. (Schematically shown in Figure 1) The AAO template has been widely applied for forming 1D nanotubes and nanowires for a range of organic materials, especially for polymers.35,36 However, to the best of our knowledge, AAO templates have never been used for preparing 0D organic nanoparticle nor any pure nanodrug of any morphology. In this work, we demonstrated for the first time that the AAO template approach can in fact be easily extended for preparing PND with much better performance comparing to the conventional reprecipitation approach. As a test case, a hydrophobic drug, teniposide (VM-26), was used as a model drug in this study. VM-26 is a typical DNAbinding agent for treating breast cancer, leukemia, lymphoma, intracranial malignant tumor, and so forth.37,38 By confining precipitation of drugs within channels of AAO templates, the size of VM-26 PNDs can be tuned via using AAO of different pore sizes. With this approach, VM-26 PNDs of sub-20 nm size are prepared for the first time and this is not achievable via reprecipitation. As structural modification of drug molecules is not needed, this simple method is demonstrated to be easily applicable for a wide range of drugs (e.g., VM-26, paclitaxel (PTX), tamoxifen (TMF), carmustine (BCNU), methotrexate (MTX), and 6-mercaptopurine (6-MP)). Moreover, the present technique enables a high production rate of 1.26 mM comparing to ∼50 μM by the conventional reprecipitation technique. B

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Figure 2. Characterization and in vitro and in vivo biomedical profiles of VM-26 pure nanoparticles. (a) SEM (inset: chemical structure of VM-26) and (b) TEM images of VM-26 NPs show uniform spherical particles, (c) TEM image of PEGylated VM-26 NPs. (d) Dynamic light scattering measurement of VM-26 NPs and PEGylated VM-26 NPs in deionized water with a diameter of 85.4 and 101.3 nm, respectively. (e) Cumulative drug release from VM-26 NPs, PEGylated VM-26 NPs, and free VM-26 molecule as a function of release time in PBS medium. (f) Confocal microscopic image showing cellular uptake of VM-26 NPs. (g) In vitro cell toxicity tests. Concentration-dependent cell survival data for A549 cells treated with PEGylated VM-26 NPs and free VM-26 molecule drugs. (h) In vivo anticancer activities of VM-26 NPs. Relative tumor volume changes with time of A549 tumor-bearing Balb/c mice after intravenous injection with PBS, free VM-26, and PEGylated VM-26 NPs. (i) Body weight variations of the A549 tumor-bearing mice indicate a negligible acute toxicity.

would increase with both AAO’s pore sizes and the concentrations of drug solutions (Supporting Information Figure S4 and S5). In the PNDs fabrication process, AAO templates were first soaked in the drug solution. The templates were then taken out of the solution and allowed full evaporation of the solvent, leaving PNDs in the pores of the AAO templates. It was observed that using a lower concentration of drug solution would lead to formation of smaller nanoparticles. We also found that the rate of solvent evaporation and solvent types have comparatively small effects on the size of PNDs. These suggest that the final size of nanodrugs primarily depend on the number of locally available molecules and external confinement of the AAO templates. To confirm that the PNDs prepared with the present approach do have the same or even better pharmaceutical effects comparing to the free drug molecules, PND of VM-26 with 70−80 nm size was selected as an example for further tests. The final drug nanoparticles have a uniform spherical shape and average diameters of ∼70 and ∼50 nm as observed with SEM (Figure 2a) and TEM (Figure 2b), respectively. The

smaller size as observed with TEM may be attributed to the vacuum drying during the TEM sample preparation as well as the Au coating during the SEM sample preparation. To enhance their biocompatibility, surfaces of the NPs were functionalized with poly(maleic anhydride-alt-1-octadecene)− polyethylene glycol (C18PMH−PEG) (Figure 2c). From Figure 2c, it can be seen that each PEGylated PND particle consists of a spherical core surrounded with a thin shell. Diameter distributions of the non-PEGylated and the PEGylated PND particles are shown in Figure 2d. It can be seen that the average diameter of the PEGylated sample (101.3 nm) is larger than that of the non-PEGylated sample (85.4 nm) by ∼15 nm, which matches well to the thickness of the thin shell as shown in Figure 2c. These confirm that the PEGylation process simply puts a thin shell onto the original PND nanoparticle (Figure 2d). For comparison, we also prepared VM-26 nanostructures by reprecipitation. It can be seen from Supporting Information Figure S6 that their morphologies and size distribution are not uniform. The as-prepared PEGylated PNDs through AAO template assisted approach have good stability in water (Supporting C

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Figure 3. (a−f) SEM images of a range of PNDs, indicating the universal application of this preparation strategy, insets are the chemical structures of different drugs: (a) 6-mercaptopurine (6-MP), (b) Paclitaxel (PTX), (c) Carmustine (BCNU), (d) Tamoxifen (TMF), (e) Methotrexate (MTX), (f) 5,10,15,20-tetro(4-pyridyl)porphyrin (H2TPyP), (g) SEM of 6-MP PNDs, and (h) VM-26 PNDs prepared by dissolving the AAO template with diluted phosphoric and citric acid, respectively, and (i) VM-26 PNDs prepared by ultrasonication. Scale bar is 200 nm. (All the images have the same magnification).

Information Figure S7) and a high drug loading capacity as determined with the ultraviolet−visible (UV−vis) absorption spectra (Supporting Information Figure S8). Due to the absence of a drug carrier, the average loading efficiency here is estimated to be 89.3%, which is much higher than that in carrier-based nanomedicines (generally less than 10%). Furthermore, a proton nuclear magnetic resonance (1H NMR) spectrum of VM-26 PNDs dissolved in deuterated chloroform and UV−vis absorption spectra of free VM-26, PNDs, and PEGylated PNDs dissolved in tetrahydrofuran show similar traces, implying that the VM-26 drug remains unchanged by the process (Supporting Information Figures S9 and S10). The drug release profile is of great importance in applying the proposed system for practical drug delivery. As depicted in Figure 2e, the remarkable increase in the release rate of the nanodrugs is attributed to the increased surface area and dissolution rate, whereas the comparative low release of free VM-26 is because of their molecular hydrophobic properties, which leads to poor water-solubility. On the other hand, due to the dissolution of the exterior PEG layer, the VM-26 NPs have a slightly slower release rate after functionalization. Cellular uptake of VM-26 NPs on A549 cells was investigated with confocal microscopy by observing fluorescence from a red dye (DCJTB) doped into the VM-26 NPs during PND preparation (Figures 2f and Supporting Information Figure S11). Strong cytoplasmic red fluorescence surrounding nuclei inside the cells was clearly observed, indicating accumulation of VM-26 NPs in cells. To investigate the possibility of utilizing the PNDs for drug delivery, we measured the cancer-cell-killing ability of the PEGylated VM-26 NPs. As depicted in Figure 2g,

the nanoparticle group shows an apparently higher cytotoxicity than the free group because of more released VM-26 molecules from PEGylated VM-26 NPs over 24 h. We also tested the cytotoxicity of surfactant C18PMH−PEG to further verify the prominent anticancer efficiency is attributed to the PNDs instead of surfactant (Supporting Information Figure S12). Encouraged by the above in vitro data, we next evaluated in vivo anticancer activities of PNDs. As shown in Figure 2h, the PEGylated VM-26 NPs treated group displays slightly stronger antitumor effect comparing to the free VM-26 control groups and the effect improves with time, possibly owing to their preferential accumulation in tumor site via the EPR effect and enhanced stability in tumor environment. We also measured the body weight of mice in each cohort. As Figure 2i presents, mice of the three treated groups show no observable differences in body weight, indicating low toxicity of the PNDs as well as no significant safety concern of potential AAO/Al residue. To demonstrate the feasibility of the present technology for practical applications, we next measured its capacity for mass production. For this purpose, we determined the concentrations of the VM-26 NPs obtained by the AAO templateassisted and reprecipitation methods (Supporting Information Figure S6d). Notably, the concentration of PND obtained via the template-assisted approach is as concentrated as 1.26 mM, which is over 25 times higher than that prepared with the conventional reprecipitation approach (∼50 μM). Photographs of PNDs solid powder by freeze-drying further demonstrate the production rate of PNDs (Supporting Information Figure S13). We also put a table in the Supporting Information (Table S1) to compare concentrations of NPs obtained from our approach and those from conventional reprecipitation methods. D

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UV−vis absorption spectra of free VM-26, VM-26 NPs, and PEGylated VM-26 NPs dissolved in a mixture of tetrahydrofuran and water, (Figure S11) time-dependent confocal microscopy images, (Figure S12) the biocompatibility of the surfactant C18−PEG measured by MTT assay, (Figure S13) digital photos of PNDs solid powder obtained by freeze-drying from PND dispersions, (Figure S14) SEM images of multiple drugs NPs, (Figure S15) schematic illustration of carrier-free PNDs via AAO template-assisted strategy for multifunctional and synergistic cancer theranosis, (Table S1) different information including material, method, concentration, mass, and theoretical maximum concentration of nanomaterials obtained from our approach and those from conventional reprecipitation methods. This material is available free of charge via the Internet at http://pubs.acs.org.

In general, the present approach would be useful for making PNDs of hydrophobic molecules. For hydrophilic molecules, it would be more difficult to obtain their corresponding PNDs as they are more prompt to erosion during the cleaning process. To demonstrate the versatility of the present approach, we further extended our works on other hydrophobic drug molecules. As shown in Figures 3a−f, we have fabricated PNDs of another six types of hydrophobic anticancer drugs using the same approach. Considering the fact that there are some other anticancer drugs can be sensitive to even very low concentration of NaOH, we further demonstrated that the AAO template can also be removed with dilute acids. PND NPs of 6-MP and VM-26 prepared by dissolving the AAO template with diluted phosphoric (1 vol %) and citric (0.677 M) acid are shown in Figure 3g and h, respectively. In fact, we found that NP of the PND can also be obtained by ultrasonication of the drug-loaded AAO template in water without dissolving the AAO template. PND NPs of VM-26 obtained by ultrasonication is shown in Figure 3i. In addition, PNDs containing multiple drugs can also be prepared, signaling that this strategy is convenient for performing combination therapy (Supporting Information Figure S14). The schematic illustration in Supporting Information Figure S15 further explains the application of making carrier-free PNDs by our AAO template-assisted strategy for multifunctional and synergistic cancer theragnosis. These results suggest that the present approach is versatile and can be easily applied for preparing NPs of single or multiple drugs even if the drugs are sensitive to diluted acid or base. To summarize, for the first time, we successfully developed a novel, versatile, and controllable strategy for preparing PNDs via an AAO template-assisted method. In comparison with the reprecipitation method, our approach produces nanoparticles with improved reproducibility and homogeneity. Mass production (1.26 mM v.s. ∼50 μM by reprecipitation) and size tunability (10−100 nm) are simultaneously achieved for VM-26 PNDs. This approach (1) provides a controllable method for obtaining PNDs of different sizes with excellent monodispersion and uniform morphology, (2) achieves mass production of nanodrugs, which plays a significant role in future clinical translations, and (3) can be easily applied to a wide range of hydrophobic biomolecules and extended for preparing all-in-one nanostructures with different functional agents. In the near future, we will systematically investigate the absorption, distribution, metabolism, and excretion of the PNDs with this approach and aim to provide more information for their potential clinical applications.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ASSOCIATED CONTENT

S Supporting Information *

Description of materials and methods and Supporting Figures: (Figure S1) cross-sectional SEM images of an AAO template, (Figure S2) the energy dispersive X-ray (EDX) spectrum of asprepared VM-26 PNDs, (Figure S3) TEM images and SAED pattern of PNDs (VM-26) with size of a) 10−20 and b) 30−40 nm, (Figure S4, S5) VM-26 NPs fabricated with AAO with different drug concentrations and different pore sizes, (Figure S6) VM-26 nanostructures prepared by the reprecipitation method, (Figure S7) average particle sizes and digital images of PEGylated VM-26 NP and VM-26 NPs, (Figure S8) the absorption spectra of VM-26, (Figure S9) 1H NMR spectra of VM-26 PNDs dissolved in deuterated chloroform, (Figure S10) E

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