The article delves into the optimization of poloxamer 407-conjugated gelatin to create pH-sensitive nanocarriers for controlled paclitaxel delivery in breast cancer treatment. It highlights the synthesis and characterization of nanogels with varying grafting ratios, evaluating their size, surface charge, critical micelle concentration, drug encapsulation efficiency, and in vitro release behavior. The study also assesses the impact of these nanocarriers on both normal and cancer cells, demonstrating their potential for targeted and efficient drug delivery. The research aims to minimize manufacturing costs while enhancing the therapeutic efficacy of paclitaxel, making it a valuable contribution to the field of nanomedicine.
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Abstract
Chemotherapy is one of the most prevalent and efficacious treatments for a wide variety of cancers; however, chemotherapeutic agents have clinically limited applications due to their low water solubility and risk of side effects. Nanomedicine can help to easily deliver hydrophobic and hydrophilic agents for cancer treatment. Here, we describe a nanocarrier system that enables the sustainable and controllable release of hydrophobic anticancer drugs, Paclitaxel, based on poloxamer 407-conjugated gelatin (GeP) copolymers. The particle size, zeta potential, morphology, and thermal stability of the nanogels were characterized. The successful synthesis of nanogels was confirmed by analyzing their chemical components. Among the GePs at different amounts of poloxamer 407, a ratio of gelatin and poloxamer (Ge:P) at 1:15 for preparation resulted in the nanogels being positive in charge, spherical in shape, and 97.84 ± 2.94 nm in hydrodynamic diameter (Dh), with optimal drug-carrying efficacy. The in vitro drug release from nanogels was accelerated in the tumor microenvironment at pH 5.5 in comparison to pH 7.4, and the drug release kinetics from nanogels were due to Fickian diffusion. Finally, the cytotoxicity assays indicated that GePs were biocompatible nanocarriers without toxicity on both normal (VERO) and breast cancer cell (MCF-7) lines, which could improve the pharmacokinetics and pharmacodynamics of paclitaxel. Overall, these results revealed an optimal ratio (1:15) of Ge:P for the synthesis of pH-responsive hybrid nanogels for sufficient paclitaxel releasement to kill MCF-7 for effective cancer treatment.
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Introduction
Breast cancer is a malignant cancer that accounts for about 30% of female cancers and is the leading cause of cancer-related mortality among them. It continues to be a significant global public health concern. Chemotherapy typically diminishes the possibility of recurrence by around 30% in specific patients. Paclitaxel (PTX), an antineoplastic drug derived from yew, was approved to treat breast cancer by the U.S. Food and Drug Administration (FDA) in 1994 [1, 2]. Nevertheless, the utilization of PTX is accompanied by some drawbacks that limit its therapeutic efficacy. For example, PTX exhibits inadequate selectivity and lacks the ability to distinguish between cancer cells and healthy cells, leading to unwanted side effects. Another practical concern regarding the therapeutic application of PTX is its extremely poor aqueous solubility and short elimination half-life due to rapid degradation in blood circulation. In order to overcome these problems, PTX delivery-mediated nanocarriers have been developed. These enhance the pharmacokinetics and pharmacodynamics of drugs so that chemotherapy can become a promising approach to eliminating cancer by targeting tumor areas while attenuating the adverse effects commonly associated with traditional PTX treatment [1, 3, 4].
Poloxamers, also known as Pluronics, are synthetic copolymers that consist of hydrophobic poly(propylene oxide) chains in the center, surrounded by two hydrophilic poly(ethylene oxide) chains, as PEO/PPO/PEO. Because of this triblock structure, poloxamers are amphiphilic, which means they can form self-assembled micelles in solutions that have concentrations higher than their critical micelle concentration (CMC) [5, 6]. Poloxamer-based biomaterials are a rapidly growing area of study in the pharmaceutical and biomedical fields. However, poloxamer-only formulations may have some drawbacks in use, especially in their toxicity aspects [7]. Therefore, an appropriate modification is necessary to maximize the efficiency of poloxamer-based drug delivery systems. Gelatin (Ge) is a well-known natural biopolymer that is obtained through the irreversible denaturation of collagen proteins. Ge is biodegradable, biocompatible, low-antigenic, low-cost, and commercially available and is utilized in various applications, including the food and pharmaceutical industries [8‐10]. Additionally, previous studies indicated that Ge possesses RGD sequences consisting of arginine, glycine, and aspartate. These RGD sequences can bind to integrins αV (particularly αVβ3), which are present at high levels on the membrane of both tumor cells and tumoral endothelial cells. In contrast, these integrins are lowly expressed on the remaining endothelial cells and most normal organs [11‐13].
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Nanogel-based drug delivery platforms have demonstrated significant promise in the advancement of anticancer therapy. Specifically, stimuli-responsive nanogels can serve as smart nanomedicines for enhancing the delivery efficiency of chemotherapeutic agents [14, 15]. Previous studies showed the potential of nanogel systems formed from gelatin and various pluronic types as efficacious carriers for the delivery of anticancer drugs, which can enhance therapeutic effectiveness and reduce side effects via sustained and selective drug release [16‐18]. Nonetheless, manufacturing scale-up is a major obstacle to the translation of nanomedicines to clinical products. It is crucial to consider minimizing the expense in the development process of nanocarriers due to the expensive price of raw materials while still ensuring superior efficiency compared to free drugs [19].
This study aimed to optimize the grafting proportion for the conjugation of Poloxamer 407 (P407) and gelatin, which forms self-assembled nanogels, to advance the delivery efficiency of paclitaxel for breast cancer as well as lessen manufacturing costs. Therefore, GePs with different amounts of P407 were prepared and characterized in terms of size, surface charge, critical micelle concentration, drug encapsulation efficiency, drug loading capacity, in vitro release behavior, and impacts on both normal (Vero) and cancer cells (MCF-7). We hypothesized that all GeP samples would be safe nanocarriers due to Poloxamer 407 being considered the least toxic member among other poloxamer copolymers, and both Ge and P407 previously received approval from the FDA [7, 20].
Materials and methods
Materials
Gelatin type A (derived from porcine skin) was obtained from Merck (Darmstadt, Germany). Poloxamer 407 (MW: 12600 g/mol), Paclitaxel, N-hydroxysuccinimide (NHS, Purity 98 %), and 1-ethyl-(3-dimethyl-aminopropyl) carbodiimide hydrochloride (EDC, Purity 98 %) were acquired from Sigma-Aldrich. p-nitrophenyl chloroformate (NPC) and 3-amino-1-propanol (Ami) were purchased from Acros Organics, Belgium. All organic solvents used, such as chloroform and diethyl ether, were analytical grade and bought from Fisher Scientific, USA. Cellulose dialysis membranes (molecular weight cut-off (MWCO): 12–14 kDa and 3.5 kDa) were supplied by Spectrum Laboratories, USA.
Preparation of poloxamer 407 grafted gelatin copolymer (GeP)
The synthesis of grafted copolymers based on Ge and P407, with four mass grafting ratios (1:5; 1:10; 1:15; 1:20 wt/wt), was performed using a multistage process as presented in Fig. 1. The initial step was the activation of P407 using NPC to obtain NPC-P407-NPC. Next, NPC-P407-NPC underwent partial replacement with Ami to generate NPC-P407-OH. This was then conjugated with Ge to synthesize an amphiphilic GeP copolymer via carbamate formation.
Fig. 1
Synthetic diagram of Poloxamer 407, Gelatin, and grafted copolymers (GeP)
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Synthesis of Ami-P407-NPC
The preparation of Mono p-nitrophenyl chloroformate-activated Poloxamer 407-Ami (NPC-P407-OH) was conducted as described previously, with slight modifications [21, 22]. Firstly, 1 mmol of P407 was magnetically stirred for 2 h under vacuum conditions at 65 °C in a double-neck round-bottom flask. Next, a quantity of 2.5 mmol of NPC was introduced into the liquefied P407. Following a 7-h reaction period, the mixture was brought to room temperature and subsequently supplemented with 30 mL of chloroform. The solution was then precipitated three times in diethyl ether at 0 °C, followed by filtration and evaporation to achieve a consistent mass, leading to the acquisition of the NPC-P407-NPC product. NPC-P407-NPC and Ami (1 mmol of each) were separately dissolved in 30 mL and 15 mL of chloroform, respectively. The Ami solution was gradually mixed with the NPC-P407-NPC solution for 12 h at room temperature. The solution obtained was then subjected to precipitation three times in diethyl ether at 0 °C. Finally, it was filtered and evaporated until a constant mass was achieved, resulting in the acquisition of the product Ami-P407-NPC.
Synthesis of GeP copolymers
The GeP copolymers were prepared following the protocol in prior studies with some adjustments [16, 17, 23]. Briefly, 100 mg of Ge was completely dissolved in 20 mL of distilled water at 40 °C in a single-neck round-bottom flask. The refrigerated Ami-P407-NPC solution was gradually introduced into the Ge solution. The mixture was magnetically stirred for 24 h at temperature not exceeding 25 °C to facilitate the formation of the amide bond (-NHCO-) and then underwent dialysis against refrigerated-distilled water using a cellulose membrane with a MWCO of 12–14 kDa for 5 days to eliminate unreactive and unwanted products. Ultimately, the GeP products were subjected to freeze-drying to obtain the GeP copolymers, which had varying grafting ratios of 1:5, 1:10, 1:15, and 1:20 wt/wt.
The GeP products were subjected to structural analysis using Fourier Transform Infrared (FT-IR) spectroscopy and Proton Nuclear Magnetic Mass Spectroscopy (1H-NMR) and the thermal properties were evaluated via thermogravimetric analysis (TGA). The TGA experiments were performed in a TGA/DSC instrument (Model: Mettler Toledo, Columbus, OH, USA), operating in a nitrogen atmosphere at a flow rate of 40 mL/minute. The dried samples (5 − 10 mg) were placed in an alumina crucible, and the experimental temperature ranged from 25 to 800 °C with a heating rate of 10 °C/minute to find out the percentage of mass loss with an increase in temperature. Simultaneously, the size distribution and zeta potential of nanogels were assessed using Dynamic Light Scattering (DLS), and the iodine probe procedure was utilized to determine their CMC values.
Preparation of Paclitaxel-loaded GeP nanogels (GeP/PTX)
For drug encapsulation, GePs (100 mg) and PTX (3 mg) were dissolved in 10 mL of distilled water and 10 mL of ethanol, respectively. Each drop of PTX solution was subsequently injected into the GeP solutions under sonication conditions, and the new mixture was then maintained under these conditions for 15 min at 10 °C. Following this, the ethanol solvent was evaporated until the sample dried and formed a thin film around the mixture container. After 5 mL of chilled distilled water (4 °C) was added, the sample was then left in a dark environment at room temperature for 24 h. Lastly, the systems were centrifuged at 5000 rpm for 10 min in order to separate the free PTX precipitates from the PTX-containing nanogel supernatant. The physical properties (such as size and surface charge), structure, and morphology of the resultants were examined using DLS and Transmission Electron Microscopy (TEM). PTX content in GePs/PTX was determined using an Agilent 8453 UV–Vis spectrophotometer (Agilent, USA) at 254 nm wavelength. The drug encapsulation efficiency (EE%) and drug loading capacity (DL%) of nanogels were calculated according to the following formulas [17, 24‐26]:
The in vitro release behavior was conducted utilizing the dialysis bag diffusion method [18, 27]. Initially, GeP/PTX samples were dispersed in 1 mL of distilled water. These solutions were placed into different dialysis bags (MWCO 3.5 kDa) and then immersed in 10 mL of PBS solution at different pH values of 7.4 or 5.5. These systems were stirred magnetically at 100 rpm at 37 °C ± 1 °C. In order to determine the amount of PTX that was released from the dialysis tube, solution samples were withdrawn at designated time intervals (0, 12, 24, 36, 48, 60, 72, 84, and 96 h), followed by measurement of PTX concentration using UV–Vis spectroscopy with absorption wavelengths at 254 nm. Concomitantly, the same volumes of fresh PBS were reintroduced into vials to prevent any variation in the volume of the media for each trial. The cumulative release rate of PTX was calculated according to the following formula [25, 27]:
In the formula, tis the amount of PTX M0is the amount of PTX present in GePs.
In order to analyze the kinetics and mechanisms of PTX release from GeP, the following six different models were applied to release data [18, 24, 25, 28]:
The mean dissolution time (MDT) value was used to assess the average time at which drugs are released from a dosage form and the impact of the polymers on the sustained release profile of the drugs. A high MDT value signifies that the drugs are released at a slower rate from the dosage form due to their effective interaction with polymeric platforms, and vice versa. In our research, MDT was calculated using the subsequent equation [18]:
where: n is the exponent and k is the release rate constant according to the Korsmeyer equation.
In vitro cytotoxic evaluation
Cell line and cell culture
The breast cancer cell line (MCF-7) and Vero cells (kidney, African green monkey) were obtained from ATCC (American Type Culture Collection, USA) and CLS (Cell Lines Service GmbH, Germany). The cells were maintained in suitable mediums, including DMEM (Dulbecco's Modified Eagle Medium), EMEM (Eagle's Minimum Essential Medium, Sigma-Aldrich, USA), or RPMI 1640 (ThermoFisher, Waltham, Germany) supplemented with L-glutamine 2 mM, antibiotics (Penicillin + Streptomycin sulfate), and 5–10% Fetal Bovine Serum (FBS) at 37 °C in a 5% CO2 humidified incubator.
MTT assays
The cellular biocompatibility of pure P407 and GeP nanogels with different amounts of P407 was evaluated on both normal and cancer cells, using the MTT colorimetric assays [27, 29]. The suspended MCF-7 and Vero cells in the cultured medium were seeded in a 96-well plate at a density of 1.5 × 105 cells/well for 24 h and then incubated with unloaded nanocarriers at a concentration of 100 µg/mL. Additionally, the cytotoxicity of PTX in both free form and loaded form (dosed at 0.05, 0.1, 0.25, 0.5, and 1.0 µg/mL) was also assessed on MCF-7 cells through the same assay. The formazan crystals, which were formed in each well after the MTT solution-adding step, were dissolved in dimethyl sulfoxide (DMSO, Sigma-Aldrich) and subsequently quantified spectrophotometrically at λ = 540/720 nm to record optical density (OD) using an Infinite F50 instrument (Tecan, Männedorf, Switzerland). The untreated group was utilized as a negative control with cell viability of 100% for the purpose of comparison. The cell viability of the samples was determined by calculating the proportion of viable cells treated by the samples compared to the control group:
The IC50 value (µg/mL or µM) is the concentration of the test sample at which 50% of cell survival is inhibited. This value was obtained using the TableCurve AISN software, with samples demonstrating at least 50% inhibition activity.
Statistical analysis
The data were presented using mean ± SEM (standard error of the mean). Differences were examined by Kruskal–Wallis and Mann–Whitney tests using Origin software. * p < 0.05 (marginally significant), ** p < 0.01 (significant), *** p < 0.001 (highly significant) were utilized to indicate statistical significance. If p ≥ 0.05, then the results are not statistically significant (expressed as ‘ns’).
All the above experiments were replicated at least three times for high reliability.
Results and discussion
Physicochemical characterization of GeP nanogels
Structural characterization of GeP nanogels
The FT-IR spectra of P407, NPC-P407-NPC, and Ami-P407-NPC are illustrated in Figs. 2(b), 2(c), and 2(d), respectively, corresponding to the characteristic valence vibrations at 3500, 3491, and 3435 cm−1 (O–H bonds); valence vibrations of the C-H bonds in the (-CH2) and (-CH3) groups of P407 were at (2970–2885), (2972–2881), and (2973–2878) cm−1; and symmetric valence vibrations of the C–O–C bonds in the P407 molecule were at 1109, 1108, and 1101 cm−1. The presence of the wavenumber at 1594 cm−1 is due to the (-NO2) group directly attached to the aromatic ring on p-nitrophenyl chloroformate and the vibration of the (C = O) group in the ester bond (-COO-NPC) of NPC [27].
Fig. 2
FT-IR patterns of a Ge, b P407, c NPC-P407-NPC, d Ami-P407-NPC, and e GePs
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The presence of NPC-P407-NPC and the urethane bond (-NHCOO-) of Ami-P407-NPC were detected at wavenumbers of 1770 cm−1 and 1643 cm−1, respectively. The presence of vibrational signals of P407, NPC, and Ami in the Ami-P407-NPC sample from the FT-IR spectrum provided evidence that the Ami-P407-NPC compound was successfully synthesized [21].
The FT-IR spectra of Ge, Ami-P407-NPC, and GePs are depicted in Figs. 2(a), 2(d), and 2(e), indicating characteristic absorption bands of (-OH) groups at wavelengths of 3423 cm−1, 3435 cm−1, and 3427 cm−1, as well as (-COC-) at 1101 cm−1 and 1109 cm−1 [17]. The first and second amide vibrations in Ge appeared at 1648 cm−1 and 1549 cm−1. For the GeP copolymers, the bands were observed at 1655 cm−1 and 1549 cm−1. Additionally, the FT-IR results of GePs revealed two new strong absorption bands at 2883 cm−1 and 1112 cm−1, corresponding to the (-CH2) and (-CO) groups on the polymer chain. The spectra obtained from FT-IR proved that GeP copolymers were successfully synthesized [23, 30].
In 1H-NMR analysis (as shown in Fig. 3), NPC-activated P407 indicated distinct resonant peaks at 1.30 and 3.36–3.75 ppm. The NPC fraction exhibited aromatic proton signals at 7.38 ppm (7) and 8.26 ppm (8). The presence of a new vibration at 4,445 ppm (5) confirmed the activation of NPC and P407 molecules [31, 32]. The chemical shift from 4.45 ppm (5) to 4.32 ppm (6) was attributed to the replacement of NPC with Ami [22], providing evidence for the successful synthesis of Ami-P407-NPC.
Fig. 3
1H-NMR spectra of a Ge, b P407, c NPC-P407-NPC, d Ami-P407-NPC, and e GePs
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For further confirmation of successful synthesis, the chemical structure of typical GeP is illustrated in Fig. 3(e). On the GeP spectrum, proton vibrations of P407 and amino acids (Leucine (Leu), Valine (Val), Isoleucine (Ile), Threonine (Thr), Alanine (Ala), Arginine (Arg), Aspartic (Asp), Lysine (Lys), …) on Ge were observed [18]. It is crucial to emphasize that aromatic protons of NPC did not exhibit any detectable signal. Furthermore, there was no signal indicating a direct bond between the methylene group and the carbonate group of NPC (-CH2-O-NPC) at 4.45 ppm (5). The only signal recorded at 4.32 ppm (6) corresponded to the methylene protons of the CH2-O-Ami bond [17, 23], confirming the conjugation of P407 with Ge.
Thermal stability of the grafted GeP copolymers
In addition to the results from FT-IR and 1H-NMR analysis, TGA was also conducted to confirm further our conclusions as well as assess the thermal characteristics of the GeP systems. The TGA curves of the tested materials are shown in Fig. 4.
Fig. 4
TGA thermogram of Ge, P407, and GePs prepared with various proportions between Ge and P407
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The TGA analysis of pure Ge revealed three different stages of mass loss within the temperature range under investigation. The initial mass loss in the range of 45–140 °C, referred to as stage 1, was attributed to the removal of water from the structure. Ge exhibits strong hydrophilicity as a result of the presence of amine and carboxylic functional groups. This characteristic allows Ge to retain a significant amount of water within its structure, in addition to the inherent presence of hydroxyl (-OH) and carbonyl (= O) groups resulting from the amino acid composition of Ge. The second mass loss began around 200–420 °C, and the final mass loss occurred above 500 °C. The mass loss observed above 200 °C indicated that the Ge structure underwent thermal denaturation. This degradation was attributed to the interactions within the Ge molecule, primarily among the amino acids that form the three-dimensional (3D) structure of Ge. The thermal treatment led to the modification or breakdown of these interactions, resulting in the observed mass loss [17]. According to the observations from the TGA results in Fig. 4, P407 exhibited a single mass loss stage, and the temperature range of this transition transpired between 300 and 410 °C. The findings obtained from the TGA analysis align with previous reports regarding the degradation of poloxamer polymers [22, 33].
The collected TGA data from GeP nanogels were divided into three distinct phases of mass losses and are shown in Table S1 in the Supplementary Information. The temperature ranges for these phases were 50–300 °C, 300–420 °C, and 420–600 °C. The temperature ranges were maintained at a constant level to make comparisons. The differences between GeP patterns and both pure Ge and P407 were also observed through the TGA curves as well as thermal parameters and the corresponding mass losses that occur during each stage of sample degradation. At temperatures exceeding 300 °C, all GeP copolymers experienced a substantial decrease in mass, which can be attributed to a major mass loss of both P407 and Ge from their compositions. In which, nanogels with proportions of 1:5 and 1:10 exhibited enhanced thermal stability due to their higher Ge content. The disparities detected between the GeP samples and P407 proved the successful integration of poloxamer onto gelatin, or more precisely, the existence of Ge within these copolymers. This presence of gelatin ameliorated the thermal stability of poloxamer, resulting in GeP systems with higher thermal decomposition temperatures than pure poloxamer. The TGA analysis revealed that P407 had a thermal degradation temperature below 420 °C, whereas both GePs and pure Ge showed breakdown temperatures over this threshold. Based on the previous analyses and the TGA results, it was confirmed that the amphiphilic GeP nanogels were prepared successfully with four different grafting ratios.
By analyzing the thermal behavior of Ge, P407, and GeP copolymers at 420 °C, where Poloxamer undergoes full decomposition, the composition of each polymer could be determined. As shown in Table S2, the constituents of copolymers varied according to the grafting proportions. Nonetheless, from a ratio of 1:15, the composition of GeP copolymers exhibited insignificant change despite a variation in Ge and P407 content.
Critical micelle concentration for nanogel formation
The stability of the drug/polymer combination is crucial in order to achieve long-term cargo delivery and maximize the accumulation of drugs in the target site without any interruptions. The physical stability of the micelle in the physiological environment is determined by two factors: thermodynamic stability and kinetic stability. Specifically, thermodynamic stability is primarily influenced by the CMC, which is the minimum concentration needed for micelle formation [34]. CMC is a crucial aspect that must be taken into account when studying drug delivery systems that utilize micelles as hydrophobic drug carriers. The CMC values of P407 and amphiphilic GeP copolymers are presented in Table 1.
Table 1
Particle size, polydispersity index (PDI), surface charge, and critical micelle concentration (CMC) measurements for GeP nanocarrier synthesis with variant Gelatin (Ge) and Poloxamer 407 (P407) proportions
Sample
Dh (nm)
PDI
Zeta potential (mV)
CMC (ppm)
Ge
/
/
25.31 ± 0.65
/
P407
35.58 ± 2.05
0.18 ± 0.05
−22.87 ± 0.21
38.61 ± 2.45
GeP (1:5)
239.17 ± 4.73
0.57 ± 0.19
15.24 ± 0.42
79.08 ± 6.24
GeP (1:10)
128.31 ± 3.27
0.42 ± 0.05
11.72 ± 0.29
76.56 ± 5.37
GeP (1:15)
97.84 ± 2.94
0.39 ± 0.05
9.55 ± 0.71
54.18 ± 4.61
GeP (1:20)
68.79 ± 2.18
0.26 ± 0.07
6.75 ± 0.22
35.36 ± 3.19
In this study, the prepared samples with varying concentrations were supplemented with a KI/I2 solution. [17]. The polymeric micelles have a core–shell structure, with a hydrophilic shell composed of PEO segments and a hydrophobic core containing PPO segments. I2 is water-insoluble, so it will be captured and accumulate within the hydrophobic core of these micelles. When the concentration of the sample is lower than the CMC, the iodine absorption demonstrates a linear pattern. Once the CMC is reached, the absorption of iodine experiences a fast rise as a result of the dispersion of I2 inside the micelles. To ensure the thermodynamic stability of GeP nanogels, the copolymer concentration must be higher than its CMC [16‐18].
Raised CMC values were observed in GeP samples compared to micelles generated from pure P407. This phenomenon may be explained by the augmentation of the hydrophilic component of the copolymer when Ge was conjugated to P407, leading to the rise in the CMC of the solutions. On the other hand, when comparing micelles formed from different P407 ratios with each other, an increase in the concentration of poloxamer was inversely proportional to the particle size, surface charge, PDI, and CMC values of the GeP copolymers. In other words, as the concentration of poloxamer was raised, these values of the GeP nanogels would decline. This relationship suggests that a higher concentration of poloxamer leads to more efficient self-assembly of the GeP copolymers into micelles due to a lower CMC value.
Cancer cell membranes have an anionic charge because of distinct sugar metabolism pathways compared to those of healthy cells. This phenomenon may be elucidated by an increased rate of glycolysis, resulting in the secretion of a substantial amount of lactic acid [35]. Consequently, nanoparticles (NPs) that have a positive charge are taken up by cells more preferentially than those with a negative charge due to electrostatic attraction. Nevertheless, cationic NPs are easily trapped by the lumina of vascular endothelial cells, which are rich in anionic phospholipids; meanwhile, highly anionic nanoparticles are susceptible to being caught by the filtration traps of the liver, spleen, or other components of the reticuloendothelial system (RES) since they resemble foreign entities like viruses and bacteria [36]. Moreover, positively charged NPs can be covered by negatively charged serum proteins, leading to opsonization, complement activation, and accelerated removal from the body. Therefore, a neutral surface charge is considered appropriate for nanomedicines in cancer therapy [37].
As presented in Table 1, the surface charge of P407 micelles was initially negative but inverted to neutral or positive in the GePs, depending on the amount of P407 grafted on Ge. This is due to charge neutralization that occurred when Ge was integrated with the PEO component of P407, forming the shell on the exterior of the nanogels. Gelatin type A is positively charged in the experimental mediums, and if the PEO blocks are less prevalent, the system will exhibit a net positive charge. When the amount of P407 was supplemented, the negative charge of P407 balanced the positive charge of Ge, leading to a neutral-charged system. The polydispersity index (PDI) is used to quantify the width of the particle size distribution, ranging from 0 to 1. PDI values below 0.5 primarily exhibit homogeneous dispersion [38, 39]. GeP nanogels were well-suspended except for the 1:5 ratio, which showed heterogeneous particle size distribution with PDI > 0.5.
Determination of drug entrapment efficiency and drug loading capacity of nanogels
The DLS results in Table 1 showed that Ge possessed hydrophilicity and electropositivity thanks to the amino (-NH2) groups, in contrast to the negatively charged P407 component, which had hydrophobic PPO segments. As a result, decreased particle sizes were observed as the P407 amounts increased based on electrostatic attraction. PTX is a hydrophobic substance with electronegative characteristics [4]. Therefore, when PTX was encapsulated into the P407 systems, the polymer molecules could mainly interact with PTX through hydrophobic interactions; in addition, electrostatic interactions also contributed to enhanced PTX-carrying efficiency. In particular, the PPO domain of P407 formed a hydrophobic space for packing PTX, while the cationic Ge shell of the GeP systems was able to load negatively charged PTX molecules because of their opposite charge.
The results of PTX encapsulation efficiency and loading capacity are presented in Figs. 5(a) and 5(b), respectively. These clearly show that Ge incorporated with P407 at the ratios of (1:15) and (1:20) would give superior drug-carrying performance compared to pure P407 and other proportions with lower amounts of P407. However, there was no statistically significant difference between these two ratios. Thus, a proportion of 1:15 between Ge and P407 was considered optimal because it could save P407 contents for nanogel synthesis.
Fig. 5
Results of varying Ge:P proportions for influencing a drug entrapment efficiency, and b drug loading capacity in GeP/PTX (n = 3). c TEM image and d size distribution histogram of GeP (1:15)/PTX. *p < 0.05, **p < 0.01, ***p < 0.001 were utilized to indicate statistical significance. Non-statistical significance was expressed as ns when p ≥ 0.05
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The morphology and size distribution of GeP (1:15)/PTX nanogels were assessed by TEM and are illustrated in Figs. 5(c) and 5(d), respectively. The nanogels exhibited a narrow size distribution, aligning with the PDI results obtained from the DLS measurement. The nanogels had spherical structures with an average uniform particle size of 42.68 ± 7.42 nm, as analyzed by the ImageJ software. There was a difference in particle size between the TEM and DLS outcomes, which was caused by different methods used to prepare measured samples [39]. TEM typically requires the deposition of a dried or fixed sample onto a copper grid, while DLS measures particles in a dispersed state in a solution, which may facilitate the swelling behavior of nanogels.
Cancerous vessels are more permeable than normal vessels since the vascular endothelium surrounding tumor tissues has loose structures [11]. NPs with a size between 50 and 150 nm are regarded as the optimal choice for passive-targeting in tumor tissue through the well-known enhanced permeability and retention (EPR) effect. This is because small-sized nanoparticles with hydrodynamic diameters of less than 8 nm can be eliminated by the kidneys into the urine, leading to a limited duration in the bloodstream. On the other hand, nanoparticles greater than 200 nm are ineffective at crossing into tumors [22, 39‐41]. Therefore, the size of the GeP nanogels, except the 1:5 ratio, was anticipated to be ideal for cancer therapy since passive targeting can raise drug content accumulation in tumor tissue.
In Vitro PTX Release kinetics from GeP nanogels
An imbalance in pH gradients is regarded as a typical feature of cancer, so the difference in pH levels between blood vessels (at pH 7.4) and solid tumors (intracellular pH 5.0–6.0) was exploited to clarify the cancer-selective drug release effects [41]. Following this, the time-dependent release kinetics of PTX, which was released continuously for up to 96 h from GeP (1:15) nanogels, were measured at two pH levels (7.4 and 5.5), as can be seen in Fig. 6(a). The release of PTX at pH 5.5 was faster than at pH 7.4, indicating that the nanogels have pH-sensitive properties. The mechanism for this pH-triggered drug release can be attributed to the protonation of ionizable groups, such as amines and carboxylic acids, on the polymer structure [42, 43]. Gelatin type A is composed of both these cationic and anionic groups, with a range of possible isoelectric point (pI) values from 7.0 to 9.5. At pH 5.5, the (-NH2) groups on the lysine and arginine residues are mostly protonated, resulting in a positive charge on the gelatin molecule [44]. Furthermore, hydrophobic interactions in the nanocore are also possibly associated with the responsiveness of pH-sensitive nanocarriers [45].
Fig. 6
a In vitro release profiles of PTX at pH 5.5 and 7.4 from GeP (1:15)/PTX, and b the remaining PTX in PBS from free PTX and PTX loaded in GeP nanogels (1:15)/PTX (n = 3). * p < 0.05, ** p < 0.01 were utilized to indicate statistical significance
×
Mathematical models were utilized to assess the sustained release performance of paclitaxel in nanogels. The results (as shown in Tables S3, S4, and Figure S) revealed that the drug release kinetics vary with pH. Based on the coefficient of determination (R2), the drug release data obtained from GeP nanogels was fitted to the Baker-Lonsdale model at pH 7.4, while the Hixson-Crowell model was employed to fit drug release data at pH 5.5. According to the theory of these models, the release of PTX from the GeP nanogels was assumed to be dependent on pure diffusion in normal physiological conditions. In contrast, the drug was hypothesized to be released from systems by an alternation in the surface area and diameter of particles in the tumor microenvironment. The Korsmeyer-Peppas model is a broadly comprehensive semi-empirical equation intended to clarify the release of drugs from polymeric systems. This is a valuable tool for investigating drug release from polymeric carriers in cases where the release mechanism is uncertain or when a combination of various release mechanisms is present. In the equation of this model, n is the exponent to depict the release kinetics mechanisms [18, 28, 46]. As presented in Tables 2, S3, and S4, both P407 micelles and GeP nanogels had n values less than 0.43, which meant that the release behavior could be explained by Fickian diffusion. In addition, PTX was released more slowly as the proportion of grafted P407 increased.
Table 2
The release parameters and dissolution time of PTX release from P407/PTX and GeP (1:15)/PTX at different pH values, fitted the drug release data to the Korsmeyer-Peppas model
PTX formulation
pH
Order of release
KKP
n
R2
MDT (h)
GeP (1:15)/PTX
7.4
Fickian
0.1631
0.3104
0.9836
81.5835
P407/PTX
7.4
Fickian
0.1670
0.3110
0.9813
74.8675
GeP (1:15)/PTX
5.5
Fickian
0.2396
0.2621
0.9742
48.3602
P407/PTX
5.5
Fickian
0.2428
0.2495
0.9706
58.1238
KKP: release rate constant; n: release exponent; R2: coefficient of determination; MDT: mean dissolution time
The MDT value of PTX from the GeP (1:15) nanogels at pH 7.4 is the longest, demonstrating the best drug retention in the materials under normal physiological conditions compared to other samples. Meanwhile, an MDT value of GeP (1:15)/PTX lower than that of pure P407 micelles in acidic conditions (Table 2) proved that this system had faster drug dissolution, speeding up the release rate in the tumor microenvironment. These release behaviors could ease patient compliance with the treatment regimen while relieving the burden of frequent administrations. The combination of different types of release mechanisms allows for targeted and sustained drug delivery to the tumor site. This can promote the therapeutic effects of paclitaxel by maintaining therapeutic drug levels over an extended period, minimizing exposure to healthy tissues, and reducing the systemic toxicity caused by off-target effects. This efficient drug delivery approach can enhance the safety profile of the treatment and facilitate patient tolerability.
The chemical stability of PTX in a PBS solution (as illustrated in Fig. 6(b)) was assessed over 96 h using UV–Vis measurement. The samples were diluted with ethanol in advance of testing at each time interval (0, 24, 48, 72, and 96 h) in order to completely dissolve any crystalline but not to chemically alter PTX. After a duration of 96 h, it was observed that around 28% of the free PTX in the PBS solution had experienced degradation. In contrast, the PTX that was packed in the GeP (1:15) nanogels exhibited a significantly inferior decomposition rate of roughly 12%. PTX can be degraded through various mechanisms, including hydrolysis, oxidation, and light-induced decomposition. These processes lead to the formation of a deteriorated PTX, which might have an altered chemical structure that potentially reduces its therapeutic activity. The outcomes of our experiments clearly showed that the GeP nanogels effectively prevented PTX from being degraded in this way, which demonstrated that this delivery system offers efficient safeguarding, improving the chemical stability of PTX.
In vitro cytotoxic studies
Cellular biocompatibility of drug-unloaded nanocarriers
Vero cells were chosen in this study because they were obtained from normal kidney cells as opposed to immortal cancer cell lines. Vero cells possess the characteristics of normal cells, including cell contact inhibition. Moreover, Vero cells are often used as a standard line for the toxicity assessment of drugs as well as biomaterials due to their efficient toxicity screening and fast growth rate with high replication potential [47‐49].
According to the MTT assays (as illustrated in Fig. 7), there was no significant cell death observed on Vero and MCF-7 cells that were cultured with the drug-unloaded GePs at a dosage of 100 µg/mL. Notably, the observed growth of both cell lines incubated with GePs showed an inverse relationship with the rise in the poloxamer component. Nonetheless, the cellular growth rate treated by GeP (1:15) was still higher than that of pure P407 groups (p < 0.05) and equivalent to that of the control group.
Fig. 7
Cellular viability of a Vero cells and (b) MCF-7 cancer cells was evaluated after 24 and 48 hours of culture with P407 alone and GePs (100 ppm) (n = 3). * p < 0.05, ** p < 0.01p, and *** p < 0.001 compared with the control group; # p < 0.05, ## p < 0.01 compared with the P407 group, were used to exhibit statistical significance. The non-statistical significance of p ≥ 0.05 was expressed as 'ns'
×
Ge is derived from collagen, which is a major component of the extracellular matrix (ECM). As a result, Ge can mimic certain aspects of the ECM and provide a more physiological environment for cells, promoting cell attachment and spreading [10]. By raising the P407 ratio, the amount of Ge present in the nanogels went down, thus reducing their ability to facilitate cell proliferation. Overall, all nanogel samples did not exhibit inhibitory effects on both healthy and cancerous cells, indicating that nanogels possess high cytocompatibility.
Anticancer efficiency of PTX-encapsulated GeP nanogels
MCF-7 cells were incubated with different materials, including various drug dosages of free PTX and PTX-loaded GeP nanogels, to compare their effects on cellular viability. The results of these assays are illustrated in Fig. 8. The cell viability of the groups exposed to free PTX drastically declined as the concentration of PTX went up, while cells that were treated with GePs/PTX progressively lost vitality because the drug slowly leaked out of the nanogels. This may be explained by the dissimilar cell uptake of free PTX and GePs/PTX. Free PTX might passively diffuse across the cell membrane due to a very steep concentration gradient, resulting in rapid cellular toxicity. On the other hand, the mechanism of cancer cell uptake for nanogels could be separated into three different stages. GePs/PTX first accumulated in tumor tissues through the EPR effect and were then internalized into cancer cells via a receptor-mediated endocytosis process based on the binding between the RGD sequence of Ge and certain integrins, such as αvβ3 and αvβ5, which are often overexpressed on the surface of various cancer cells. In the final stage, the encapsulated drugs were released in the tumor microenvironment, where the conditions are relatively acidic in comparison with normal tissues [11, 50, 51].
Fig. 8
The cell viability of MCF-7 breast cancer cells was examined by free PTX and GePs/PTX for one-day cultivation (n = 3). * p < 0.05, ** p < 0.01, and *** p < 0.001 were used to denote statistical significance. Non-statistical significance was represented as 'ns' when p ≥ 0.05
×
After 24 h of incubation, the cytotoxicity of free PTX was very high, with an in vitro half-maximum inhibitory concentration (IC50) of 6.79 ± 1.2 nM. Nevertheless, when GeP (1:15) nanogels were utilized to carry PTX, the toxicity was dramatically decreased, with an IC50 value of 38.96 ± 2.9 nM (presented in Table S5). The results in Table S6 revealed that the IC50 values of GeP (1:15)/PTX nanogels gradually went down over time. This demonstrated that paclitaxel was efficiently carried within nanogels and then released in a controlled manner, as well as accumulating in the tumor site after cellular internalization. The combination of these outcomes and the biocompatible tests above also confirmed that the cytotoxicity of PTX-loaded GeP nanogels within the experimental concentration range was completely due to the released PTX.
Conclusions
In summary, an attractive paclitaxel-delivery platform, poloxamer 407-modified gelatin nanogels, was successfully synthesized and characterized. The hydrodynamic size range of nanogels was from 68.79 to 239.17 nm, depending on the amount of conjugated poloxamer. GeP nanogels at the ratio of 1:15 demonstrated appropriate physicochemical characteristics, a significant increase in both drug encapsulation efficiency and drug loading capacity, and were proven to deliver PTX in a controlled and targeted manner associated with pH-sensitive properties. At a concentration of 100 μg/mL, GeP nanogels showed high cell viability and non-toxicity to both normal and cancer cells. Additionally, using GePs as the drug carriers, in vitro anti-breast cancer results exhibited the gradual and sustainable cytotoxicity of PTX over time. Based on these findings, it was verified that the grafting proportion of 1:15 could serve as an optimal option for manufacturing GeP nanogels, which efficiently administer PTX and offer a promising approach in the field of controlled drug delivery systems for cancer chemotherapy. Therefore, further in vivo studies should be conducted to evaluate the prospect of this delivery system in more detail, which will be helpful for clinical application purposes. Interestingly, 3D cell culture approaches, such as multicellular spheroids, organoids, microfluidics, and 3D bio-printing, can mimic the biological behavior of cancer cells. These in vitro models should be considered before executing animal experiments and human clinical trials in the final stage.
Declarations
Conflicts of interest
The authors declare that there are no competing interests that are relevant to the content of this article.
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