Breast cancer is the most commonly detected cancer [1
] in women with 1,700,000 new cases worldwide. Almost 883,000 new cases were identified in developing countries along with 794,000 new cases in developed countries, making the disease the fifth common reason for women’s death per year [2
]. The morbidity and mortality rate are high due to the distant metastasis which involves cancer cell detachment from the primary site of the tumor, entrance into the systemic circulation and tumor cell proliferation in the other organs parenchyma [5
]. Hence it is important to deliver the drugs in a targeted manner for the suppression of breast cancer cells metastasis or invasiveness.
The Raf/MEK/ERK signalling pathway (Raf = rapidly accelerated fibrosarcoma, MEK = MAPK/ERK kinase, ERK = extracellular signal–regulated kinases) is recognized as the mitogen-activated protein kinase (MAPK) cascade comprising guanosine-nucleotide-binding protein RAS (GTPase) which stimulates Raf-family proteins including A-Raf, b-Raf and c-Raf/Raf1 [6
]. This signalling cascade controls cell proliferation, differentiation, metastasis, survival, and angiogenesis. RKIP (Raf-kinase inhibitor protein) is a master modulator of the Raf/MEK/ERK signalling pathway, which can interfere with the Raf-1 facilitated phosphorylation and activation by interrupting the interaction between the MAP-kinase and the MAP-kinase activated extracellular signal-regulated kinase (ERK) [1
]. However, RKIP expression is highly reduced in lymph node metastasis of breast cancer [7
The impaired outcome of the Raf/MEK/ERK signalling cascade may cause the incidence of different types of tumorigenesis [6
]. As such, Raf-1 controls the anti-apoptotic transcription factor, nuclear factor-κB as well as caspase-8 activation. Again, overexpression of Raf-1 may cause multi-drug resistance phenotype [8
]. In contrast, b-Raf is more commonly mutated than A-Raf and c-Raf [9
] in a human tumor, promoting tumour cell proliferation and survival by elevating kinase activity and stimulating downstream MEK–ERK signaling [10
]. Hence it is a crucial treatment strategy to inhibit activation of b-Raf mutation, resulting in downregulation of the MEK kinase and preventing the cancer cell growth.
Hydrophobic in nature, AZ628 is a type II selective and potent pan-Raf kinase inhibitor under clinical pipeline by AstraZeneca. It impedes anchorage-dependent and -independent growth, resulting in the arrest of the cell-cycle, and induces apoptosis in different cancer cell lines [9
]. By inhibiting the activity of preactivated b-Raf, b-Raf V600E and c-Raf, it stops MEK activation and sustains sensitivity to the MEK inhibitor, thus causing effective inhibition of cancer cell proliferation [10
The targeted delivery of a hydrophobic anticancer drug is a challenging process due to its solubility issue. Owing to hydrophobicity, the chemotherapeutics are not readily soluble in the aqueous environment of the body and tissue fluids. Moreover, the hydrophobic anticancer drugs are prone to aggregation upon intravenous administration, leading to embolism and local cytotoxicity [12
]. Nanoparticles (NPs) present an auspicious approach to address the lipophilicity issue of a hydrophobic drug and can improve drug accumulation in tumor sites by altering the biodistribution and pharmacokinetic properties [4
]. Since NPs are substantially larger in size than the anticancer drugs, they cannot cross the tight junctions between endothelial cells of the healthy blood vessel. This phenomenon generally prohibits the NPs from carrying the chemotherapeutics inside the healthy tissue. The tumour site is, on the other hand, composed of the leaky vasculature and reduced number of weakened lymphatic drainage, thus enabling the right sized NPs to enter the site with long-term accumulation. This phenomenon, known as the enhanced permeability and retention (EPR) effect, is the basic concept for a passive tumour targeting [12
Broadly speaking, NPs can be grouped into organic and inorganic nanomaterials. Depending on the physicochemical properties, inorganic nanomaterials are usually prefered over organic ones [16
], mostly because of the easier formulation process, controlled particle size distribution, targeted delivery of the drug, stability and rapid release of the payload [17
Carbonate apatite (CA) Ca10
is a smart non-viral pH-sensitive inorganic carrier owing to its biodegradability, heterogeneous surface charge, and limited crystal growth. CA NPs possess PO43−
-rich and Ca2+
-rich domains in their outer surfaces that provide a broader scale of binding opportunity for both positively and negatively charged drugs through ionic interactions. From the systemic circulation, the drug-loaded CA NPs can enter into the cancer cell via endocytosis, resulting in particle degradation and discharge of the encapsulated drug inside the endosomal acidic microenvironment [20
]. However, CA NPs tend to form larger particles by aggregation process [22
], which can be stabilized by the addition of serum albumin [23
] or biotin-PEG [17
Krebs cycle compounds such as sodium citrate, sodium succinate and α-ketoglutaric acid are natural, inexpensive, harmless and environment-friendly [24
]. In our previous study, we found that the incorporation of citrate and succinate in CA NPs played a pivotal role in modulating the particle size, cellular uptake, amount of drug binding and cytotoxicity, depending on the number of carboxylic acid groups in the salt structures [4
]. Here, α-ketoglutaric acid, an intermediate of Krebs cycle was used to transform the CA particles into α-ketoglutaric acid-modified CA (α-KAMCA) NPs. The effects of such modification on the particle size, drug-loading efficiency, and enhancement of cellular uptake and cytotoxicity of AZ628 were also examined.
3. Materials and Methods
3.1. Materials and Chemicals
DMEM (Dulbecco’s modified eagle medium) powder was procured from Gibco by Life Technology (Thermo Fisher Scientific, Waltham, MA, USA). Sodium bicarbonate (NaHCO3), calcium chloride (CaCl2.2H2O), and alpha-ketoglutaric acid disodium salt hydrate salts were purchased from Sigma-Aldrich (St Louis, MO, USA). Anti-cancer drug AZ628, DMSO (Dimethyl sulphoxide) and thiazolyl blue tetrazolium bromide (MTT) were obtained from Sigma–Aldrich (St Louis, MO, USA). DMEM-high glucose liquid media, FBS (Fetal Bovine Serum), TrypLE Express and penicillin-streptomycin were acquired from Sigma–Aldrich (St Louis, MO, USA). Acetonitrile (ACN) was from Fischer Scientific (Loughborough, UK). Bradford 1× dye reagent was purchased from Bio-rad Laboratories (Hercules, CA, USA).
3.2. Fabrication and Turbidity Measurement of CA and α-KAMCA NPs
DMEM-buffered solution was prepared by dissolving 0.675 g DMEM powder with 0.185 g NaHCO3
in 50 mL Milli Q water and 1 N hydrochloric acid was used to fix the final pH of the solution to 7.4 [21
]. CA NPs was generated by mixing 4 mM of calcium chloride dihydrate in 1 mL of freshly prepared DMEM solution and the final mixture was incubated at 37 °C for 30 min. For optimizing the particle formation, alpha-ketoglutaric acid disodium salt hydrate at 1, 2, 4, 8 and 16 mM was dissolved separately along with 4 mM exogenous Ca2+
in 1 mL of DMEM media and incubated all the combination at 37 °C for 30 min to formulate α-KAMCA NPs. After 30 min incubation, the turbidity of CA and α-KAMCA NPs were assessed at 320 nm wavelength through a UV-visible spectrophotometer (Jasco, Oklahoma, OK, USA).
3.3. Turbidity Measurement of Alpha-Ketoglutaric Acid Salt
The turbidity of alpha-ketoglutaric acid salt dissolved in DMEM media was checked to analyze any possible interference of the salt with the turbidity of the formulated α-KAMCA NPs in 320 nm wavelength. Thus, α-ketoglutaric acid salt at 1, 2, 4, 8 and 16 mM was added to 1 mL DMEM solution and incubated at 37 °C temperature for 30 min. All other conditions for the particle formation were maintained in the same manner and the turbidity of the salt in the DMEM media was measured.
3.4. Optical Images of α-KAMCA Particle Formation
The images of aggregated particles were taken by Olympus Fluorescence Microscope IX81 (Shinjuku, Tokyo, Japan) with 10× magnification at a scale bar of 50 µm. α-KAMCA particles were formed by mixing alpha-ketoglutaric acid salt at 0, 1, 2, 4, 8 and 16 mM concentrations with 4 mM Ca2+ and incubating for 30 min at 37 °C. After the particle formation, all the particle suspensions were transferred to a 24-well plate and optical images were taken immediately.
3.5. Estimation of Drug Encapsulation Efficacy
The high-performance liquid chromatography method was performed to estimate the concentration of the drug encapsulated in the NPs. The standard curve was prepared by plotting the concentration of the drug (AZ628) vs. the peak area. The drug was used at 0, 20, 40, 60, 80 and 100 µM concentrations to get the peak area. AZ628 at 60 and 100 µM concentration was added with 4 mM exogenous calcium in 1 mL DMEM media to create AZ628-bound CA NPs. Similarly, AZ628 at 60 and 100 µM concentration was mixed with 4 mM exogenous Ca2+ and 4 mM alpha-ketoglutaric acid salt in 1 mL DMEM solution to produce AZ628-loaded α-KAMCA NPs. The mixture of salts and drug was then incubated at 37 °C for half an hour. Next, the drug-particle suspension was centrifuged at 13,000 rpm for 30 min at 4 °C using a Refrigerated Bench-Top Microcentrifuge (Eppendrof, Hamburg, Germany). After the particle precipitation, the supernatant was collected and the amount of the drug present in the supernatant was checked by Agilent chemostation software attached with HPLC (Agilent, Santa Clara, CA, USA). To perform this experiment, zorbax C18 column (4.6 × 150 mm, Agilent, Santa Clara, CA, USA) was used and the mobile phase was ACN and Milli Q water in a proportion of 82.5:17.5 (v/v), pumped at a constant flow rate of 1.0 mL/min. Quantification was performed at DAD (Diode Array Detector) wavelength of 254 nm.
The standard curve (y
+ 19.214, R2
= 0.9981) was used to calculate the concentrations of AZ628 present in the supernatant.
The % drug encapsulation efficiency was calculated using the following formula:
] drug bound with NP
’ is the concentration of AZ628 entrapped in NPs calculated from the standard curve and ‘[M
] initial drug conc.
’ is the total concentration of AZ628 used to run HPLC (or initially mixed for the preparation of AZ628-loaded NPs formulations), “[N
] unbound drug conc.
” is the concentration of free AZ628 in the supernatant (the amount of drug that is not bound with the NPs), “[P
] peak area of supernatant
” is the area of unbound AZ628 in the supernatant detected by HPLC.
where “Mass of AZ628 bound to NPs
” is the amount of AZ628 entrapped in NPs (μg/mL) and “Mass of NPs recovered
” is the number of NPs collected after centrifugation (μg/mL). The experiments were performed in triplicate and presented as average ± SD (standard deviation).
3.6. Size and Surface Charge Measurement
For the size and surface charge measurement, AZ628-loaded α-KAMCA NPs were generated by adding the drug at 1, 10, 100 and 1000 nM concentrations, together with 4 mM Ca2+ and 4 mM alpha-ketoglutaric acid salt to 1 mL of freshly prepared DMEM. AZ628 in 1 nM to 1 µM concentration with 4 mM Ca2+ was added to 1 mL freshly prepared DMEM media to formulate AZ628-loaded CA NPs. In a similar way, drug-free α-KAMCA particles were prepared by mixing alpha-ketoglutaric acid salt at 1 mM to 16 mM strength with 4 mM exogenous Ca2+ to 1 mL of freshly prepared DMEM solution. CA NPs was formulated by mixing 4 mM of Ca2+ in 1 mL of freshly prepared DMEM solution. All the mixtures of salts were incubated at 37 °C for 30 min, after which 10% FBS was added to all the preparations. The experiment was performed without any dilution of the samples. During measurement, all the preparations were kept in the 4 °C ice chiller. Malvern Nano Zetasizer (Malvern, Worcestershire, UK) was used to measure the particle size (z-average) in diameter, the particle size by intensity (dynamic light scattering) and the zeta potential.
3.7. Culture and Seeding
The human breast cancer cell line, MCF-7 cells, and the mouse breast cancer cell line, 4T1 cells, were cultured in two separate 25 cm2 flasks with a complete DMEM (cDMEM) media (pH 7.4) containing 10% FBS, penicillin and streptomycin antibiotic and the flasks were placed in a humidified incubator at 37 °C with 5% CO2. Both cell lines were collected from the exponential growth phase, subjected to trypsinization process, following repeated washing through centrifugation steps and seeded on a 24-well plate (Greiner, Frickenhause, Germany). Each of the wells contained approximately 50,000 cells and was subjected to overnight incubation prior to treatments.
3.8. Fabrication of NPs and AZ628-Loaded NPs and Cell Treatment
DMEM solution was freshly prepared according to the above-mentioned protocol and the pH of the final media was adjusted to 7.4 using 1 N HCl. AZ628 at 0.1, 1, 10, 100 and 1000 nM concentrations was added along with 4 mM calcium and 4 mM alpha-ketoglutaric acid salt to 1 mL of filtered DMEM solution. Likewise, AZ628 in 0.1, 1, 10, 100 and 1000 nM strength was added with 4 mM Ca2+ to 1 mL filtered DMEM media. These mixtures were then incubated for 30 min at 37 °C to generate AZ628-loaded α-KAMCA NPs and AZ628-incorporated CA NPs. The same concentrations of AZ628 were added to 1 mL DMEM solution and incubated in the same manner to prepare free drug solutions as a control. Drug-free α-KAMCA NPs were also fabricated by the addition of alpha-ketoglutaric acid salt at 1 mM to 16 mM concentrations with 4 mM Ca2+ in DMEM media. The complete DMEM medium in each well was replaced with 10%-FBS-supplemented medium containing either free AZ628, nanocarriers alone, or AZ628-loaded nanocarriers. After that, the treated 24-well plates were kept inside the incubator for 48 h until the MTT assay was performed. The similar treatment was prepared to perform MTT assay at 24 h time point to evaluate time-dependent cytotoxicity in MCF-7 cell line.
3.9. MTT (3-(4,5-dimethlthiazol-2-yl)-2,5-diphenyltetrazolium Bromide) Assay in a Different Cell Line
MTT assay was performed for the estimation of cytotoxicity of the free drug, free NPs and drug-loaded NPs in MCF-7 and 4T1 cell lines after 24 h and 48 h of treatment. 50 μL of MTT (5 mg/mL in PBS) was added to each well of the plates which were subsequently incubated for 4 h at 37 °C in the incubator humidified with CO2 (5%) to convert into formazan crystals. Next, 300 μL of DMSO solution was added to each well after the removal of the MTT medium. After 5 min, the plates were shaken vigorously on the microplate reader to solubilize the purple colored formazan crystals in DMSO. The absorbance of dissolved formazan crystals was measured spectrophotometrically by using a microplate reader (BIO-RAD-Microplate Reader) at a wavelength of 595 nm with reference to 630 nm. The experiments were performed in triplicate and presented as average ± SD.
3.10. Experimental Investigation
The cell viability in percent was quantified by using the values extracted from the MTT assay:
= the absorbance of the treated cells and C
= the absorbance of the control.
The toxicity (%) of the free drugs and drug-loaded NPs in cancer cell line was calculated, respectively, by using the following formula:
is the cell viability (%) of the control, CVfree AZ628
is the cell viability (%) of free drug and CVNP bound-AZ628
is the cell viability (%) of NPs bound drugs.
The enhanced cytotoxicity (%) of AZ628-loaded NPs was assessed using the following formula:
= Absorbance of the sample treated with NPs, P
= Absorbance of the sample treated with free AZ628, Q
= Absorbance of the control
= % cell viability of the control (100%), A
= % cytotoxicity of α-KAMCA NPs, B
= % cytotoxicity of free AZ628 and D
= % cell viability of AZ628-loaded α-KAMCA NPs. The experiments were performed in triplicate and presented as average ± SD.
3.11. Cellular Uptake in MCF-7 and 4T1 Cell Lines
Free AZ628, AZ628-loaded CA and AZ628-loaded α-KAMCA NPs prepared with 40 µM and 100 µM of the drug. The freshly prepared treatment was used to treat MCF-7 (~5 × 106 cells) and 4T1 cells (~5 × 106) to evaluate the dose-dependent cellular uptake for AZ628. The supernatant of culture media was collected after 4 h of the treatment and centrifuged at 13,000 rpm for 30 min at 4 °C. The resultant supernatant was subjected to HPLC to determine the amount of drugs present in the supernatant (representing the free drug not taken up by the cells).
Finally, the cellular uptake was calculated by using the following formula:
= Initial drug concentration, FC
= Free drug concentration in the supernatant.
The experiments were performed in triplicate and presented as average ± SD.
3.12. Western Blot
MCF-7 cells (~5 × 106
cells) were seeded and incubated at 37 °C in a humidified atmosphere with 5% CO2
for 24 h. After that, the seeded cells were treated with CA NPs, α-KAMCA NPs, CMCA NPs, AZ628-loaded CA NPs, AZ628-loaded α-KAMCA NPs, AZ628-loaded CMCA NPs and free AZ628 for 24 h. CMCA NPs were formulated according to the protocol of our previous study [4
], however in the current study only CA NPs and α-KAMCA NPs were discussed. After treatment with the free drug, free NPs, and drug-loaded NPs, the cells were incubated for 24 h, followed by the cell detachment and cell lysis at 4 °C by using a lysis reagent (protease inhibitor, phosphatase inhibitor, sodium fluoride & stable stock lysis buffer). The lysed cells were centrifuged at 13,000 rpm for 30 min at 4 °C to collect the proteins from the supernatant. Quick Start Bradford Protein Assay kit (Bio-Rad) was used in estimating the total protein contents in the cell lysates. About 10 μg of total proteins were loaded for SDS-PAGE in stain-free Mini protein TGX gels (Bio-rad, USA) in the 1× running buffer at 50 V for 2 h. Proteins were transferred to a nitrocellulose membrane (Bio-rad, Germany) by wet blotting at 0.35 A for 2 h and the membrane was blocked with 5% skimmed milk in tris-buffered saline along with Tween-20 (1× TBST) for 1 h at room temperature. The membrane was incubated overnight with primary antibodies (T-MAPK, T-Akt, p-MAPK, p-Akt, Caspase-3 and GAPDH as an internal control) at 1:1000 dilutions at 4 °C with gentle shaking. The membrane was washed with TBST (5 × 10 mL) for 5 min each and further incubated with a secondary antibody (Thermo Fisher Scientific, Waltham, MA, USA) at 1:3000 dilution for 1 h at room temperature with gentle shaking. After the incubation with secondary antibody, the membrane was washed with TBST (5 × 10 mL) for 5 min to remove any unbound antibody. The membrane was incubated with a mixture of luminol and peroxide Clarity Western ECL substrate (Bio-rad, USA) at room temperature for 5 min. Protein bands were visualized using Chemidoc XRS Imaging system (Bio-rad, Hercules, CA, USA) following the manufacturer’s protocol.
α-KAMCA NPs and CA NPs were prepared according to the protocol described above and centrifuged at 13,000 rpm for 30 min and the supernatant was removed. The pellets were re-suspended in 1 mL Milli-Q water prior to centrifugation at 13,000 rpm for 30 min. Finally, 30 µL Milli- Q water was added to the pellets after the removal of the supernatant. The sample was placed on a sample holder coated with carbon tape and left to dry at room temperature. After that, the samples with subjected to platinum sputtering for 40 s with 30 mA sputter current at 2.30 tooling factor. The morphology and size of the samples were visualized at 5.0 kV and 2.0 kV using FE-SEM (Hitachi/SU8010, Tokyo, Japan).
3.14. Biodegradability Profiles of α-KAMCA NPs, AZ628-Loaded α-KAMCA NPs and AZ628-Loaded CA NPs
α-KAMCA NPs were generated using 20 mM Ca2+ and 20 mM alpha-ketoglutaric acid salt in 200 µl PCR tubes. AZ628 at 25 µM, exogenous calcium at 20 mM and α-ketoglutaric acid salt at 20 mM were added to 200 µl of freshly prepared DMEM solution to formulate highly concentrated AZ628-loaded α-KAMCA NPs. AZ628 was added in 25 µM strength with 20 mM exogenous Ca2+ to 200 µL of freshly prepared DMEM solution. All the mixtures were incubated at 37 °C for 30 min to generate the NPs (with or without the loaded drug). After that, the suspended NPs were added to 800 µL of DMEM media at different pH ranging from 7.5 to 5.0. Next, the turbidity of the NPs suspensions of different pH was measured at 320 nm by a UV-VIS spectrophotometer. The experimental results of three replicates were presented as average ± SD.
3.15. Stability test of NPs
CA NPs were formulated using 4 mM exogenous Ca2+ in 1 mL freshly prepared DMEM media and subjected to incubation at 37 °C for 30 min. Likewise, α-KAMCA NPs were generated by mixing 4 mM α-ketoglutaric acid with 4 mM Ca2+ in 1 mL of DMEM media and placed inside an incubator of 37 °C for 30 min. Ten percent FBS was added to each formulation. After the formulation, the turbidity of the NPs was measured at 320 nm wavelength using a UV-visible spectrophotometer at different timepoints, 0, 0.5, 1, 2, 4, 8, 24, 48 and 72 h to check the stability of CA and α-KAMCA NPs. Throughout the process CA and α-KAMCA NPs were kept at 37 °C.
3.16. Statistical Analysis
Statistical significance was analyzed in drug-treated versus drug-loaded NPs-treated groups by one-way ANOVA, followed by post-hoc analyses using the Scheffe multiple comparisons test (SPSS version 23 for Windows). The minimal level of statistical significance was p < 0.05 with 95% confidence interval (CI). The experiments were performed in triplicate and presented as average ± SD.
Different parameters such as diverse concentration gradients of salts, changed pHs of the media, altered incubation time and temperature, and partial replacement of phosphate with carbonate [20
] and Ca2+
with a divalent cation such as magnesium (Mg2+
] or strontium (Sr2+
] have been highly studied to improve the growth kinetics of apatite-based NPs. The uniqueness of these NPs is the pH sensitivity towards a tumor acidic environment and the ability to release the drugs inside the endosomes after internalization [20
We previously reported pH-sensitive citrate-modified CA (CMCA) and succinate-modified CA (SMCA) NPs for the delivery of Doxorubicin, a hydrophilic anthracycline chemotherapeutics to the human breast cancer cell line [4
] with promising drug binding affinity, enhanced cellular uptake, and efficient cytotoxicity. CMCA and SMCA, prepared by modifying CA with three carboxyl groups-containing citrate and two carboxyl groups-containing succinate, respectively, has inspired us to explore the physicochemical properties and drug delivery efficacy of CA NPs modified with α-ketoglutarate which contains one ketone group and two carboxylic groups.
α-KAMCA NPs were prepared by incubating at 37 °C for 30 min in a high glucose medium containing fixed concentrations of inorganic phosphate (PO43−
), calcium (Ca2+
) and bicarbonate (HCO3
) ions, with different concentrations of alpha-ketoglutaric acid salt. The formation of NPs was ensured by performing turbidity analysis (Figure 1
A) and optical image analysis (Figure 2
). The consistent increase in turbidity for α-KAMCA NPs were noticed with increasing concentration of the α-ketoglutarate salt, which clearly indicates the formation of an increased number of particles with an increase in the amount of ketoglutarate salt added. The optical image analysis demonstrated that the addition of 1–4 mM concentration of ketoglutarate salt accelerated formation of the particles; however, the number was significantly reduced at 8–16 mM concentrations of the salt. The larger particles formed at 8 mM–16 mM concentration of alpha-ketoglutaric acid was correlated with the gradual rise in turbidity of the particles. At higher concentration (8–16 mM), α-ketoglutaric acid might have the tendency to aggregate which was further supported by optical image analysis (Figure 2
F,G) and DLS analysis (Figure 4
The enhanced therapeutic efficacy is an essential prerequisite for the engineered nano-drug delivery system, which can be achieved by increasing the circulation half-life of the drug, reducing drug accumulation in the healthy tissues and by releasing the payload into the targeted tumor site [12
]. The unique size-dependent properties of nanomaterials do not allow them to cross the endothelial tight junctions of the healthy vascular lining, however, the NPs can simply cross the leaky tumor vasculature through the EPR effect which also relies on defective lymphatic drainage for prolonged accumulation in the tumour site [42
]. Three principal factors usually determine this multi-step process—the increased relative surface area, nano-scale size and surface charge of the nanomaterials [43
]. Higher EPR effect can be produced with particles of average size of 20 nm to 1 µm by prolonging systemic circulation, avoiding opsonization and reducing the uptake rate of MPS (mononuclear phagocyte system) [43
]. The average diameter for α-KAMCA NPs formulated with 4 mM ketoglutarate salt was found to be within 250–300 nm range (Figure 1
C) with monodisperse PDI value 0.322 (Figure 4
D) and therefore, would be a strong candidate for passive targeting of tumor based on the EPR effect. The zeta potential for CA and α-KAMCA NPs was between −9 to −13 mV, implying no significant difference in the surface charge between the two different NPs (Figure 1
In this work, a hydrophobic drug AZ628 was incorporated into CA NPs and α-KAMCA NPs. The drug-particle interaction was verified by drug loading capacity, surface charge of the resultant complex and its effect on cytotoxicity in different cancer cell lines. The fabricated AZ628-loaded CA NPs and α-KAMCA NPs showed average diameters of approximately 456–591 nm (Figure 3
A) and 366–479 nm (Figure 3
B), respectively. Drug-loaded NPs showed the concentration-dependent increase in particle size proportionally with the increased drug concentration (Figure 3
A,B). The surface charges for AZ628-incorporated apatite-based NPs remained same between −9 to −13 mV (Figure 3
AZ628 could bind with CA and α-KAMCA NPs by ionic interactions of its protonated amine groups in bicarbonate-buffered media of pH 7.4. Drug binding to the NPs was measured by quantifying the amount of unbound drug through HPLC assay (Figure 8
). As shown in the result, AZ628 exhibited dose-independent binding affinity towards CA and α-KAMCA NPs. At 60 µM concentration of the drug (Figure 8
B), α-KAMCA revealed 4% more binding affinity than CA NPs.
The dissolution profiles of CA NPs and α-KAMCA NPs were assessed at six different pHs of the same buffered media ranging from pH 7.5 to pH 5.0 (Figure 9
). In case of apatite-based nanocarriers, pH-responsive drug release is crucial in the target site to avail the substantial toxicity to the cancer cells while lessening the adverse effects to healthy tissues, and to overcome the multidrug resistance [48
]. In this study, drug release was indirectly measured by analyzing pH sensitivity of the NPs at the acidic environment through turbidity test. Under normal physiological conditions α-KAMCA NPs, AZ628-loaded CA NPs and AZ628-loaded α-KAMCA NPs were stable; however, the phosphate and carbonate ions in the apatite structure could readily accept excess H+
ion from the endosomal acidic microenvironment, causing the particles to be dissolved [52
] and enabling AZ628 to passively diffuse from endosome to cytoplasm with the result of enhanced drug accumulation in the tumor region and improved therapeutic efficacy.
AZ628-loaded α-KAMCA NPs demonstrated excellent MAPK and Akt downregulation in the human breast cancer cell lines, reducing proliferation and finally inducing apoptosis as reflected from the activation of cleaved Caspase-3 (Figure 11
). Further, it can be explained by the greater cellular uptake of AZ628-loaded α-KAMCA NPs and AZ628-loaded CA NPs than the free drug (Figure 8
). The α-KAMCA-facilitated cellular uptake for AZ628 was almost 57% at 100 µM concentration in both cell lines at 4 h of treatment. α-KAMCA NPs showed 25% and 34% more cellular uptake than free AZ628 at 40 and 100 µM concentration, respectively (Figure 8
). The enhanced cellular uptake clearly correlates with the decrease in cell viability (Table 2
and Table 3
). Thus, the cytotoxic effect of AZ628-loaded α-KAMCA NPs was found to be notably higher than that of the free AZ628, which could be due to the smaller particle size of α-KAMCA NPs, which provide a larger folded surface area to adsorb the sufficient amount of the drug and promote effective cellular internalization via endocytosis [20
]. Additionally, the subsequent release of AZ628 from CA and α-KAMCA NPs as a result of fast dissolution of the internalized NPs at acidic pH (Figure 9
) contributed to the enhancement in cytotoxicity.