Multi-drug delivery system based on alginate/calcium carbonate hybrid nanoparticles for combination chemotherapy

Highlights

• A facile strategy to prepare nano drug carriers for combination chemotherapy was developed.

• Alginate/CaCO₃ hybrid nanoparticles for co-delivery of multiple types of drugs were prepared.

• Both hydrophobic and hydrophilic drugs can be loaded in the nanoparticles efficiently.

• Dual drug loaded nanoparticles exhibited significantly enhanced tumor cell inhibitory effect.

• The nanoparticles have promising applications in combination chemotherapy.

Abstract

A facile strategy to prepare nano-sized drug carriers for co-delivery of multiple types of drugs in combination chemotherapy was developed. Inorganic/organic hybrid alginate/CaCO₃ nanoparticles were prepared by co-precipitation in an aqueous solution under very mild conditions. A hydrophilic drug (doxorubicin hydrochloride, DOX) and a hydrophobic drug (paclitaxel, PTX) were co-encapsulated in the hybrid nanoparticles. For comparison, PTX loaded nanoparticles and DOX loaded nanoparticles were also prepared. The measurement based on dynamic light scattering indicated all nanoparticles had a mean size less than 200 nm with a relatively narrow size distribution. The morphology of the nanoparticles was observed by TEM. The in vitro drug release study showed that the release of DOX and PTX from the dual drug loaded nanoparticles could be effectively sustained. The tumor cell inhibitory effect of the drug loaded nanoparticles was evaluated in HeLa cells and MCF-7/ADR cells. The dual drug loaded nanoparticles exhibited significantly enhanced cell uptake and nuclear localization as compared with the single drug loaded nanoparticles. As a result, the dual drug loaded nanoparticles had a significantly enhanced cell inhibitory effect, especially for drug resistant tumor cells. These results indicated that alginate/CaCO₃ hybrid nanoparticles have promising applications for the co-delivery of drugs with different physicochemical properties in combination chemotherapy to overcome multidrug resistance.

Introduction

Nanoparticles for biomedical applications, such as chemotherapy, gene delivery, diagnosis, hyperthermia therapy and phototherapy, have undergone extensive investigations in recent years [1]. Among the various applications, the nanoparticles for anti-cancer drug delivery have attracted increasing research interest in the hope of improving the efficacy of chemotherapy and reducing related side effects. Nano-sized drug delivery systems possess advantages including the convenience in administration, the passive targeting ability to tumor tissues by enhanced permeability and retention (EPR) effect, and the capability to realize active targeting through incorporation of ligands [2], [3], [4], [5].

In cancer treatments, combination chemotherapy to combine the drugs with different mechanisms of action can achieve combinative effects and minimize side effects [6], [7], [8]. As a result, co-delivery of multiple drugs has drawn growing interest in recent years [9]. For example, a nanogel based on heparin-pluronic conjugate was used to encapsulate paclitaxel and deoxyribonuclease to achieve a dose-dependent synergistic cytotoxicity [10]. D-alpha-tocopheryl-co-poly(ethylene glycol) 1000 succinate (TPGS) based nanoparticles were used to load cisplatin, docetaxel and herceptin for the treatment of breast cancer. To stabilize the nanoparticles and to facilitate herceptin conjugation, poly(lactic acid)-TPGS and carboxyl group-terminated TPGS were added in the polymeric matrix to fabricate the co-delivery system [11]. The polymersomes prepared by diblock copolymer PEG-PTMBPEC with an acid-labile poly(2,4,6-trimethoxybenzylidenepentaerythritol carbonate) (PTMBPEC) block were able to co-deliver hydrophobic paclitaxel and hydrophilic doxorubicin hydrochloride by using the water-filled core to encapsulate the hydrophilic drug and the lamellar bilayer to entrap the hydrophobic drug [12]. Polymersomes based on PEG-PLA and PEG-butadiene could co-load doxorubicin and paclitaxel for in vivo delivery [13]. Using the micelles prepared by folate conjugated PLGA-PEG and cell-penetrating peptide incorporated PLGA-PEG to co-deliver doxorubicin and paclitaxel could achieve synergistic cell inhibition effects [14]. An amphiphilic phospholipid-mimicking prodrug of camptothecin could form stable liposome-like nanocapsules with the capability to load water soluble drugs such as doxorubicin hydrochloride, and the nanocapsules could be used for co-delivery of camptothecin and doxorubicin hydrochloride [15].

Most commonly, the widely adopted methodologies for fabricating synthetic polymer based nanospheres for drug delivery involve the use of organic solvents, which may cause unfavorable toxic effects. In addition, in order to satisfy the delivery requirements of various therapeutic agents with a wide range of properties, the carriers should have specifically designed structures resorting to complicated synthesis procedures. To develop a facile method to fabricate drug carriers with good biocompatibility, which can efficiently load and deliver both hydrophobic and hydrophilic drugs simultaneously, is highly desirable.

Among different drug carriers, the carriers based on inorganic compounds including calcium carbonate (CaCO₃) and calcium phosphate (CaP) have unique advantages due to their ideal biocompatibility and the ability for loading different therapeutic agents [16], [17], [18], [19], [20], [21], [22]. Although CaCO₃ and CaP possess favorable properties for co-delivery of various drugs, the great hindrance for them to form a useful drug delivery platform is the crystallization of the compounds, i.e., the initially formed small particles tend to transform into big crystals, which are unfavorable for drug loading and delivery. According to previous studies, the presence of hydrophilic polymers could retard the crystallization and improve the colloidal stability of the CaCO₃ and/or CaP based nanoparticles [19], [20]. For example, the heparin/CaCO₃/CaP nanoparticles prepared by the co-precipitation in the presence of heparin exhibited a well-controlled size and a good colloidal stability [20].

In this study, we aim to develop a facile strategy to prepare nano-sized multiple drug delivery systems for combination chemotherapy. In our previous work, alginate/CaCO₃/DNA nanoparticles were prepared for gene delivery and alginate/CaCO₃/DNA/DOX nanoparticles were prepared for gene/drug co-delivery [23]. Since the negatively charged DNA with phosphate groups could bind Ca²⁺ ions, CaCO₃/DNA co-precipitates prepared at a Ca²⁺/CO₃²⁻ ratio of 50/1 were capable of encapsulating and transferring genes into targeted cells effectively. The modification of alginate resulted in improved stability and gene delivery efficiency. In the current study, we further prepared nano-sized alginate/CaCO₃ hybrid nanoparticles for multiple drug delivery. The alginate/CaCO₃ nanoparticles were prepared by the precipitation of calcium carbonate at a Ca²⁺/CO₃²⁻ ratio of 1/1 in the presence of alginate. The presence of alginate could offer good control over the morphology and the size of the nanoparticles. All components in the nano-carriers (CaCO₃ and alginate) have good biocompatibility and biodegradability. In addition, the pH-dependent dissolution of CaCO₃ component favors intracellular drug delivery.

To evaluate the drug loading and release properties of the alginate/CaCO₃ nanoparticles, doxorubicin hydrochloride (DOX) as a hydrophilic drug and paclitaxel (PTX) as a hydrophobic drug were co-loaded in the nanoprticles. DOX is a commonly used anti-cancer drug in clinical chemotherapy with a broad spectrum anti-cancer effect. Through acting on DNA or RNA intracellular, DOX obstructs cell proliferation by inhibiting the biosynthetic process of nucleic acids [24]. PTX is a highly hydrophobic anti-cancer drug, which inhibits cellular mitochysis by interfering with the normal breakdown of microtubules during cell division [25]. DOX combined with PTX is used as first-line treatment for metastatic breast cancer [26].

In the current study, we demonstrated both DOX and PTX could be easily loaded in alginate/CaCO₃ hybrid nanoparticles, and the nanoparticles could effectively sustain the drug release. Compared with free drugs, the drug loaded nanoparticles exhibited significantly enhanced tumor cell inhibitory effects, especially for drug resistant tumor cells. The hybrid nanoparticles prepared in this study are promising drug carriers for combination chemotherapy in cancer treatments.