In vitro and in vivo applications of alginate/iron oxide nanocomposites for theranostic molecular imaging in a brain tumor model

Abstract

Nanocomposites composed of highly biocompatible and safe alginate and iron oxide nanoparticles have been employed to encapsulate doxorubicine for brain tumor therapy. The antitumor activity of nanocomposites was demonstrated using in vitro and in vivo tests. The results significantly indicated that the nanocomposites had great safety and potential for brain tumor therapy.

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Gliomas are the most aggressive and infiltrative brain tumor, but no therapeutic methods can efficiently cure them. The average survival time of a patient diagnosed with a glioma is less than 15 months.¹ Many anticancer drugs have been found to kill glioma cells in vitro, but their antitumor activities are largely limited in in vivo tests due to their poor solubility and short half-life in circulation. Combining targeted therapy with nanoparticle (NP)-based drug delivery can overcome these critical obstacles because of that Drug/NP complexes can prolong the circulation time in the blood and increase the local concentration of drugs in the tumor region.² However, the blood-brain barrier (BBB) provides a natural shield for the brain against the invasion of various toxins and restrict from passage only to necessary substrates from the circulation to the brain tissue.³ Thus, the BBB also limits the brain uptake of diagnostic and therapeutic agents, which results in lower therapeutic efficiency.⁴ Using BBB-disruption strategies to allow therapeutic agents to enter the brain is not suitable, because it may also allow circulating toxins in the blood to enter the brain.³ Brain is more important than other tissues, so the used agents are highly required to be safe and cannot damage and affect brain functions. Therefore, an extensive and urgent search for real safe and effective platforms that can non-invasively deliver therapeutic drugs across the BBB to specifically kill glioma cells is under way.

Many studies of using NPs to increase drug delivery to the brain have been proved, and they showed different delivery efficiencies.⁵ The base requirement of BBB-penetrating therapeutic agents is they must not damage the brain or cause adverse side effects. The ideal components of nanocomplexes must be biocompatible, biodegradable, and non-cytotoxic. Currently, most used carriers to cross the BBB are various functionalized liposomes.⁶ However, the size control and stability of liposomes are problematic. Gold NPs are often used treating brain diseases because of their excellent biocompatibility and low cytotoxicity. Gold NPs decorated with peptides⁷ or insulin⁸ as BBB-penetrating platforms have also been reported. However, gold-based nanomaterials lack the detection ability in magnetic resonance imaging (MRI), a powerful modality for detecting the details of brain regions and diagnosing brain diseases. To mitigate this drawback, gadolinium (Gd³⁺) chelates^7,9 or iron oxide NPs¹⁰ are usually used to conjugated with gold-based nanomaterials to allow for MRI. In fact, the synthesized processes of functionalized gold nanocomplexes for MR applications are complicated and inconvenient. Thus, using magnetic nanomaterials as primary nanocarriers to directly allow MRI is better choice. For future clinical development, using materials that are already permitted by the U.S. Food and Drug Administration (FDA) to develop therapeutic agents is a better strategy due to they had completed clinical data and trials. However, the in vivo applications of nanomaterials composed of high safe or FDA-approved materials for brain tumors are still limited.

Here we report the development of a nanocarrier composed of alginate and Fe₃O₄ NPs and mainly focus on its safety and therapeutic efficiency. This nanocarrier can encapsulate an anticancer drug, doxorubicin (Dox), to treat brain tumor, both in vitro and in vivo (Scheme 1). Importantly, Fe₃O₄ NPs and alginate are highly safe, biocompatible and biodegradable materials, and they are permitted by the U.S. FDA for use in humans. Both materials had a wide variety of pharmaceutical, biomedical, biotechnology and tissue engineering applications. Moreover, the metabolites of Fe₃O₄ and alginate are also safe in human bodies and don't cause side effects.

Scheme 1 (a) Schematic synthesis process of biosafe anticancer drug-encapsulating nanocarriers. (b) Schematic mechanism of Dox-encapsulating nanocarriers and then Dox release from nanocarriers to brain tumor region to kill tumor cells.

The NH₂-exposed Fe₃O₄ NPs (NH₂-Fe₃O₄ NPs) were synthesized using the co-precipitation method previously described.¹¹ Subsequently, alginates were conjugated with NH₂-Fe₃O₄ NPs using a covalent bond on the particle surface. The true diameters of NH₂-Fe₃O₄ NPs and alginate-conjugated NH₂-Fe₃O₄ NPs (alg-Fe₃O₄ NPs) are separately calculated as 6.3 nm and 6.6 nm in transmission electron microscopy images (Fig. S1a†). However, hydrodynamic diameter is preferred to be used in biomedical applications, so the hydrodynamic diameters of NH₂-Fe₃O₄ NPs and alg-Fe₃O₄ NPs were measured as 19.2 nm and 138.6 nm by dynamic light scattering (DLS) instrument (Fig. S1b†). The discrepancy in hydrodynamic diameter of alg-Fe₃O₄ NPs is due to the existence of alginate on the surface of NPs, compared to NH₂-Fe₃O₄ NPs. The surface charge of the NH₂-Fe₃O₄ NPs was +20.3 mV. After conjugation with alginate, the surface charge of the alg-Fe₃O₄ NPs was −45.1 mV, which was contributed by COO⁻ groups of alginate. The change of surface charge and the discrepancy in hydrodynamic diameter of alg-Fe₃O₄ NPs indicated that alginate had been successfully tagged on the surface of NH₂-Fe₃O₄ NPs. The alginate on the Fe₃O₄ NP surface was also investigated using Fourier transform infrared spectroscopy (FT-IR) (Fig. S2†). The FT-IR spectrum of alg-Fe₃O₄ NPs involves both characteristic adsorption peaks of Fe₃O₄ NPs and alginate. The alginate content in the alg-Fe₃O₄ NPs was then determined by thermogravimetric analysis (TGA). A weight loss of about 12.1% was observed for the alg-Fe₃O₄ NPs (Fig. S3a†), corresponding to the conjugated amount of approximately ∼0.15 mg alginate per mg of Fe₃O₄ NPs.

To fabricate the Dox-encapsulated alg-Fe₃O₄ (Dox/alg-Fe₃O₄) NPs, Dox and alg-Fe₃O₄ NPs were mixed, and then 1 mM of calcium ion (Ca²⁺) aqueous solutions were added to the mixture. When mixing Dox and alg-Fe₃O₄ NPs, alg-Fe₃O₄ NPs spontaneously captured Dox by electrostatic force due to the positive charges of Dox and the negative charges of alg-Fe₃O₄ NPs. The surface alginate of alg-Fe₃O₄ NPs was physically crosslinked by Ca²⁺ to form net structures, and then Dox was trapped inside the NPs. The morphology of Dox/alg-Fe₃O₄ NPs was shown in Fig. S1a.† The saturated encapsulated amounts of Dox in Dox/alg-Fe₃O₄ NPs were ∼1.42 mg Dox per mg of Fe₃O₄ NPs (Fig. S3b†). To evaluate the Dox-trapped efficiencies of Dox/alg-Fe₃O₄ NPs, the Dox leaching of Dox/alg-Fe₃O₄ NPs were tested in deionized water and phosphate buffered saline (PBS) (10 mM, pH 7.4) at 37 °C (Fig. S4a†). The leaching percentages of Dox in both conditions in Fig. S4a† are below 10% after 240 h. These results indicate that Dox efficiently trapped in the Dox/alg-Fe₃O₄ NPs. Fig. S4b† shows the Dox release profiles of Dox/alg-Fe₃O₄ NPs in PBS (pH 5.5) and cytoplasm mimicking (CM) buffer at 37 °C. The Dox-release rate of Dox/alg-Fe₃O₄ NPs in CM buffer was faster than in PBS (pH 5.5) because of the ethylenediamine tetraacetic acid (EDTA) in the CM buffer. EDTA can strongly grab the Ca²⁺ chelated with the alginate of the Dox/alg-Fe₃O₄ NPs and destroy the crosslink structures.

To determine what were safe doses of Dox/alg-Fe₃O₄ NPs for cell and animal experiments, human umbilical vein endothelial cells (HUVECs) were incubated with different iron doses alg-Fe₃O₄ NPs at 37 °C for 24 and 48 h. The alg-Fe₃O₄ NPs showed no apparent cytotoxicity and the HUVECs had viability rates >95% (Fig. S5†). Subsequently, after 24 h of incubation with alg-Fe₃O₄ NPs, the cell viability of C6 brain cancer cells was >95% (Fig. 1). To evaluate the efficacy of Dox/alg-Fe₃O₄ NPs in cancer cells, C6 cells were separately treated with Dox and Dox/alg-Fe₃O₄ NPs for 24 h. Cell viability was significantly lower in Dox/alg-Fe₃O₄ NP-treated C6 cells than in free-Dox-treated C6 cells at all Dox concentrations because the nanocarriers increased the amount of drugs delivered to the cells and then increased the drug dose inside the cells. After 48 h of treatment with Dox/alg-Fe₃O₄ NPs, cell viability was even lower according to statistical analysis (P < 0.1). Detail safety of alg-Fe₃O₄ NPs was evaluated by determining the activities of liver enzymes (glutamate oxaloacetate transaminase [GOT], glutamic-pyruvic transaminase [GPT], total bilirubin [T-Bil], and alkaline phosphatase [ALP]) and kidney enzymes (blood urea nitrogen [BUN], creatinine [CREA], and uric acid [UA]) of mice with treatment with alg-Fe₃O₄ NPs (Fig. S6†). The levels of GOT, GPT, T-Bil, and ALP of alg-Fe₃O₄ NP-treated mice have no obvious difference compared to those of PBS-treated mice (control group) (Fig. S6a†). No apparent changes were also obtained in the levels of BUN, T-Bil, and UA of alg-Fe₃O₄ NP-treated mice (Fig. S6b†). It represents no cytotoxic effects are toward the liver and kidney functions of alg-Fe₃O₄ NPs-treated mice. Similarly, the histological studies of alg-Fe₃O₄ NP-treated mice also had no change (Fig. S7†) and indicated alg-Fe₃O₄ NPs were safe for use in clinical applications.

Fig. 1 In vitro cell viability of C6 cells incubated with free Dox, alg-Fe₃O₄ NP, and Dox/alg-Fe₃O₄ NPs at 37 °C for 24 and 48 h. All experiments were repeated in triplicate. Asterisks indicate statistically significant different of both experimental sets. (*, P < 0.1; **, P < 0.01).

The efficacy of Dox/alg-Fe₃O₄ NPs was next evaluated in an in vivo animal model by measuring tumor growth and body weight in mice with C6 tumors (∼50 mm³), which were divided into four treatment cohorts (PBS, free Dox, alg-Fe₃O₄ NPs, and Dox/alg-Fe₃O₄ NPs) (Fig. 2). All the mice were intratumorally injected with one of the four treatments. The Dox dose for each Dox-treated mouse was 3 mg kg⁻¹. The equivalent Fe dosage of Dox/alg-Fe₃O₄ NPs was 5 mg kg⁻¹ when the injection dose of Dox was 3 mg kg⁻¹. Thus, the injection doses of the control groups (free Dox and alg-Fe₃O₄ NPs) were 3 mg kg⁻¹ of Dox and 5 mg kg⁻¹ of Fe, respectively. The growth of C6 tumors was not inhibited in the PBS, alg-Fe₃O₄ NPs, or free Dox groups compared with the Dox/alg-Fe₃O₄ NPs group (Fig. 2). However, tumor growth was significantly inhibited in Dox/alg-Fe₃O₄ NPs group. The primary reason for this outcome is that C6 cells can uptake Dox/alg-Fe₃O₄ NPs, and then most of their Dox is released in the cytoplasm and then enters the cell nuclei to cause cell apoptosis. Conversely, C6 cells prevented free Dox from entering. There were no significant differences in mean body weights of the four groups of mice (Fig. S8†), which showed none of the treatments caused negative side effects. To investigate the possible therapeutic effects of future clinical applications using Dox/alg-Fe₃O₄ NPs for in vivo human brain cancer, the U87MG-luc2 human glioblastoma cell line, which expresses luciferase, was selected to establish our animal model of U87MG-luc2 tumor-bearing mice, which were divided into four treatment-based cohorts: PBS, Dox, alg-Fe₃O₄ NPs, and Dox/alg-Fe₃O₄ NPs. The alg-Fe₃O₄ NPs, Dox/alg-Fe₃O₄ NPs, free Dox, and Dox/alg-Fe₃O₄ NPs were intratumorally injected into the tumor site, and then the tumor size was monitored pre-injection and at 3 and 7 days post-injection using the IVIS image system. Tumors continuously grew in PBS-treated mice. In the mice treated with Dox and alg-Fe₃O₄ NPs, the tumor sizes showed no obvious variations until 7 days post-injection. Comparatively, tumors significantly shrank by about 50% in the mice treated with Dox/alg-Fe₃O₄ NPs. It seems that releasing the Dox directly inside mice, most of the Dox remained outside the tumor cells and had no anti-tumor effect. In order to achieve the objective of anti-tumor therapy, the mice with smaller tumors (∼25 mm³) were injected with Dox/alg-Fe₃O₄ NPs under the same experimental conditions and procedures as shown in Fig. 3. The U87MG-luc2 tumors had almost disappeared on post-injection day 3 (Fig. S9†). On post-injection day 7, the tumor had totally disappeared from the backs of some mice. This indicates that tumors smaller than 25 mm³ can be completely remitted after treatment with Dox/alg-Fe₃O₄ NPs with a Dox dose of 3 mg kg⁻¹. Thus, we hypothesize that the Dox/alg-Fe₃O₄ NPs can be used for brain tumor therapy in future clinical trials.

Fig. 2 In vivo anti-tumor activity of Dox/alg-Fe₃O₄ NPs. The tumor volume of mice in all groups were measured every day. The mice were intratumorally injected with PBS (10 mM, pH 7.4), alg-Fe₃O₄ NPs, Dox only, or Dox/alg-Fe₃O₄ NPs. The injected Dox dose was 3 mg kg⁻¹ of body weight, and the equivalently injected Fe dose was 5 mg kg⁻¹ of body weight (n = 5).

Fig. 3 In vivo anti-tumor activity of Dox/alg-Fe₃O₄ NPs in mice with U87MG-luc2 tumors ∼50 mm³ during the experimental period. All images are luminescence images from U87MG-luc2 cells and monitored using the IVIS imaging system. The mice were intratumorally injected with PBS (10 mM, pH 7.4), alg-Fe₃O₄ NPs, Dox only, and Dox/alg-Fe₃O₄ NPs. The injected Dox dose was 3 mg kg⁻¹ of body weight and the equivalently injected Fe dosage was 5 mg kg⁻¹ of body weight (n = 5).

In summary, a novel nanocomposite composed of highly safe and US-FDA-approved Fe₃O₄ NPs and alginate has been synthesized and they could encapsulate Dox inside the particles. The Dox leaching percentage of Dox/alg-Fe₃O₄ NPs is quite low in deionized water and PBS, but Dox can be released from Dox/alg-Fe₃O₄ NPs inside the tumor cells after cellular uptake. Both in vitro and in vivo experimental results showed that the Dox/alg-Fe₃O₄ NPs inhibited C6 tumor cell growth and killed them without damaging healthy non-tumor cells. Moreover, U87MG-luc2 tumor-bearing mice with larger and smaller tumors (∼50 mm³ and ∼25 mm³) are being designed to test the anti-tumor activity of Dox/alg-Fe₃O₄ NPs. Based on our results in this study, we are designing and proceeding with additional animal experiments for primary brain tumor therapy by inducing U87MG-luc2 tumor in mice brain to establish glioblastoma models. The BBB-permeating NPs based on Dox/alg-Fe₃O₄ NPs are expected to develop. The BBB-permeating NPs will be intravenously injected into mice with U87MG-luc2 tumors to demonstrate that these BBB-permeating NPs will cross the BBB and provide efficacious anti-brain-tumor therapy.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5ra20723a

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