Calcium carbonate CaCO recognized as a naturally biomineral
Calcium carbonate (CaCO3), recognized as a naturally biomineral, is
highly desirable for biomedical applications due to excellent bio-compatibility and biodegradability. Other merits like low price and ease of mass production also make CaCO3 suitable in the delivery of drugs, genes or other biomolecules [21,22]. In addition, CaCO3 is pH-labile which can dissolve in the tumor acidic environment [21,23,24]. How-ever, the CaCO3 wrapping will make the nano-vehicles easy aggregation in physiological condition ascribing to its hydrophobicity . Al-though further modification of other hydrophilic molecules (Poly-ethylene glycol (PEG), polyvinyl alcohol (PVA), dextran, chitosan, etc.) can improve nanoparticles colloid stability, they still encounter passive immune system clearance and low accumulation in targeted site . Compared with these synthetic polymers, cancer Glycols polyethylene membranes with some specific proteins (e.g. CXCR4, E-Cadherin, PD-L1, integrin, focal adhesion kinase, and RHO family proteins) which are responsible for immune escape and homologous aggregation of cancer cells, will be more suitable for camouflaging nanoparticles, because this top-down approach would extensively simplify the processes for the multi-functional decoration. Meanwhile, the self-recognition in cellular levels can lead to the eﬀ ;ective targeting of nanoparticles to the homologous tumors [27–31].
In this study, we introduced a novel CaCO3 capped MSN to realized acidic tumor microenvironment controlled drug release for cancer therapy. MSNs with a uniform size of 100 nm were prepared and then loaded with DOX. Afterwards, the drug loaded MSN was capped with a layer of CaCO3 as gatekeeper (DOX/[email protected]) for regulating DOX release. Finally, DOX/[email protected] was further camouflaged with cancer cell membrane to obtain the nanomedicine of DOX/ [email protected]@CM. The collapse and shedding of CaCO3 from MSN at acidic tumor environment was demonstrated by TEM observation. Consequently, negligible premature release in the simulated physiolo-gical environments (pH 7.4), and a quick release under tumor acidic environment (pH 6.5 and pH 5.0) was also documented. Benefiting from homotypic targeting abilities of cancer cell membrane, DOX/ [email protected]@CM was easily internalized by cancer cells and accu-mulated into tumor site, which ultimately exerted significantly en-hanced antitumor eﬀect, as demonstrated both in vitro and in vivo.
2. Material and methods
Hexadecyltrimethylammonium chloride (CTAC), triethanolamine (TEA) and tetraethylorthosilicate (TEOS) were purchased from Sinopharm Chemical Reagent Co.,Ltd (Shanghai, China). Doxorubicin hydrochloride (DOX) was bought from Sigma (USA). RPMI-1640 medium, fetal bovine serum (FBS) and trypsin-2.5% (w/v) EDTA so-lution were obtained from Gibco (USA). Mem-PER™Plus Membrane Protein Extraction Kit was supplied by Thermo-Fisher (USA). Cell Counting Kit was purchased from TransDetect. Hoechst 33342 and AnnexinV-FITC/PI apoptosis assay kit were purchased from Thermo-Fisher (USA). Live/Dead Cell Staining Kit was purchased from Biovision (USA).
2.3. Synthesis of MSNs and loading of DOX
CTAC (2 g) and TEA (0.02 g) were sequentially added into 20 mL of water. The aqueous solution was stirred at 80 °C for 1 h before dropwise addition of 1.5 mL of TEOS. After further stirring at this temperature for
1 h, the obtained mixture was centrifuged. The resulting nanoparticles were thoroughly washed with ethanol to remove the unreacted re-actants. The obtained product was then placed in a solution of NaCl in methanol (1 wt%) for 3 h to remove the CTAC. The mesoporous silica nanoparticles (MSNs) were obtained after repeating the above extrac-tion procedure for three times.
For loading of DOX, 5 mg of MSNs was mixed with 5 mL of DOX (0.5 mg/mL) in water. After stirring for 24 h, DOX-loaded MSNs (DOX/ MSNs) were collected by centrifugation and washed with water for three times. The loading content was determined to 4.2%, through measuring the absorbance of DOX in the collected supernatant solution at the wavelength of 490 nm by a microplate reader (SpectraMax M5e).
Cyclohexane (7.5 ml), Triton X-100 (1.77 ml) and 1-hexanol (1.6 ml) were mixed thoroughly. Then 400 μL of calcium chloride so-lution (30 mM) and 40 μL of sodium carbonate solution (2.92 M) were added sequentially. Finally, the mixture was further stirred overnight and then centrifuged to collect nanoparticles.
2.5. Preparation of cell membrane debris
The cracked cancer cell membrane of LNCaP-AI cells were extracted by using membrane protein extraction kit. Typically, LNCaP-AI cells were washed by Cell Wash Solution supplied by manufacturer, before suspended in the Permeabilization Buﬀer. Afterwards, this suspension was stirred at 4 °C for 10 min, and then underwent centrifugation at 700 g for 5 min. The resulted supernatant containing cracked cell membrane was collected after centrifugation at 16000 g for 15 min. Finally, the precipitated cell membranes (CM for short) were lyophi-lized.