Nanotechnology paves ways for the design and development of advanced drug delivery systems having control and ultra-precision over the release of drugs, which dramatically change the novel strategies of drug delivery. Various approaches, such as encapsulation, targeting molecules or specific biomarkers, and using artificial nanocarriers are adopted to ensure controlled and targeted (or smart) drug delivery. Biodegradable and intelligent polymeric materials pave the way for controlled, targeted drug delivery in modern therapeutics owing to their unique physical and chemical properties, excellent bioavailability, controlled release, biocompatibility, and less toxicity [33, 34]. This research aimed to design and development of thermo/pH dual responsive noncovalent graft copolymer micelles based on the host–guest interaction for enhanced drug delivery applications.
Synthesis and characterization of guest polymers
The guest polymers were prepared in three sequential steps, as explained in Scheme 1. In the first step, PHEMA as the backbone, was synthesized by the RAFT polymerization, using 4-cyano-4-(phenylcarbonothioylthio)pentanoic acid as a chain transfer agent. In the second step, PHEMA-g-PCL was synthesized by ring-opening polymerization of CL in the presence of PHEMA as a macroinitiator and Sn(Oct)2 as a catalyst. In the last step, PHEMA-g-PCL-OTs was obtained by tosylation of PHEMA-g-PCL in the presence of TEA, and then, guest copolymer benzimidazole-containing (PHEMA-g-(PCL-BM)) was synthesized via replacement of the tosyl moiety with BM in the presence of TEA. The chemical structures of the guest polymers were characterized by FTIR analysis (Additional file 1: Figure S2).
The synthesized PHEMA, PHEMA-g-PCL, and PHEMA-g-PCL-BM copolymer were further characterized using 1H NMR spectroscopy (Fig. 1B). In the 1H NMR spectrum of the PHEMA (Fig. 1B1), the proton peak at 7.4 –7.93 belonged to aromatic protons of the RAFT agent. The methylene and methyl protons of the PHEMA skeleton were appeared at 1.77–1.92 and 0.77–0.93 ppm, respectively. The protons of the –OCH, and –CHOH groups were observed at 3.88 and 3.58 ppm, respectively. Moreover, the chemical shift at 4.81 ppm was attributed to the hydroxyl groups of the PHEMA. The average degree of polymerization (\(\overline{\mathrm{DP} }\) n) of the synthesized PHEMA was determined to be ~ 69 by 1H NMR data (Additional file 1: Figure S2.3a).
A typical 1H NMR spectrum of the copolymer PHEMA-g-PCL with the assignment is shown in Fig. 1B2. The major resonance peaks (a–d) were attributed to PCL segments in PHEMA-g-PCL. The methylene proton signal (d′, δ = 3.6 ppm) indicated that PCL was terminated by hydroxyl groups. The feed molar ratio of [CL] to [OH] of PHEMA was 25:1. The average degree of polymerization for the PCL segments in PHEMA-g-PCL copolymers was calculated by 1H NMR from the integration of the signals of methylene protons (peak d, δ = 4.1 ppm) to the terminal methylene protons (peak d′, δ = 3.6 ppm) (see Fig. 1A). The molecular weight of the PHEMA-g-PCL (\(\overline{\mathrm{M} }\) n ~ 166,500 g mol−1) was determined from the 1H NMR data (Additional file 1: Figure S2.3(b)). The chemical structure of PHEMA-g-(PCL-BM) copolymer was characterized by 1H NMR, as displayed in Fig. 1B3. The major resonance peaks at 7.1–7.7 ppm corresponded to benzene protons, and the chemical shift at 8.4 ppm attributed to imidazole proton were completely appeared, which showed the benzimidazole molecules were conjugated onto PHEMA-g-PCL [35]. The results proved the successful synthesis of PHEMA-g-(PCL-BM) copolymer. The coupling efficiency of PHEMA-g-(PCL-BM) was calculated to be 70% based on the relative integral values of the methylene signal on the PCL (–CH2OCO–, 3.9–4.1 ppm) and that connected with benzimidazole (–CH2CH2–Nbenzimi, 3.6 ppm) from the 1H NMR spectra (see Eq. (4), Additional file 1: Figure S2.3(c)) [36]. In addition, the number average molecular weight of the synthesized PHEMA-g-(PCL-BM) was calculated from the 1H NMR data (Additional file 1: Figure S2.3(c)).
Synthesis and characterization of host polymers
The dual responsive host polymer β-CD-star-(PMAA-b-PNIPAM) was prepared through three sequential steps as shown in Scheme 1. In the first step, the PMAA homopolymer was synthesized through RAFT polymerization of methacrylic acid monomer using AIBN as initiator and 4-cyano-4-(phenylcarbonothioylthio)pentanoic acid as a chain transfer agent at 75 °C. In the second step, to synthesize of PMAA-b-PNIPAM copolymers, RAFT-synthesized PMAA was used as a macromolecular RAFT agent and AIBN as initiator at 85 °C. In the last step, for the synthesis of β-CD-star-PMAA-b-PNIPAM, the esterification reaction of β-CD with PMAA-b-PNIPAM was carried out in the presence of DCC and DMAP at room temperature. The success of chemical synthesis of the PMAA, P(MAA-b-NIPAM), and β-CD-star-(PMAA-b-PNIPAM) could be confirmed from the FTIR spectral results (Additional file 1: Figure S2.2), which were further supported by 1HNMR spectra.
The synthesized PMAA, PMAA-b-PNIPAM, and β-CD-star-PMAA-b-PNIPAM copolymer were further characterized through 1H NMR spectroscopy (Fig. 1C).
In the 1H NMR spectrum of the PMAA, the proton peaks at 0.81–1.14 ppm (a), 1.6–1.9 ppm (b) are assigned to the methyl and methylene protons of the PMAA, respectively (Fig. 1C(1)) [37]. In addition, the aromatic protons of the RAFT agent and hydroxyl group give peaks at 7.4 –7.93 ppm (ph) and 12.4 ppm (c), respectively [38]. The average degree of polymerization of the synthesized PMAA was determined to be ~ 21 by 1H NMR data (Additional file 1: Figure S2.3(d)).
The 1H NMR spectrum of PMAA-b-PNIPAM was shown in Fig. 1C2. The proton peaks at 0.73–1.17 ppm (d), 1.6–2.13 ppm (a), and 2.18–2.3 ppm (b) in the 1HNMR spectrum of PMAA-b-PNIPAM copolymer corresponded to the methyl, methylene, and methine groups existing on the carbon skeleton of the copolymer. Moreover, the –CH–NH protons of the PNIPAM block (c) were observed at about 3.86 ppm. \(\overline{\mathrm{DP} }\) n of PNIPAM was calculated by comparing the composition of PNIPAM block with PMAA block using the integral area of their respective protons, as indicated in Fig. 1C (see Eq. (8), Additional file 1: Figure S2.3(e)). \(\overline{\mathrm{M} }\) n, NMR of PNIPAM was calculated, and molecular weight of the prepared PMAA-b-PNIPAM diblock copolymer was obtained by combining \(\overline{\mathrm{M} }\) n, NMR of PMAA and PNIPAM blocks (see Fig. 1A). The molecular weight of the PMAA-b-PNIPAM was determined to be 5480 g mol−1 (Additional file 1: Figure S2.3(e)).
The 1H NMR spectrum of the β-CD-star- PMAA-b-PNIPAM was shown in Fig. 1C3. After the reaction, the new peaks at 2.5 ppm (g), 3.2−3.6 ppm (h, e, f), 4.4 ppm (d), 4.8 ppm (c), and 5.5−5.8 ppm (b) are assigned to the β-cyclodextrin group. According to the integral of C(k)H and C(c)H, the number of carbonyl groups is calculated as ~ 7 per β-CD molecule. The molecular weight of the β-cyclodextrin-star-PMAA-b-PNIPAM was calculated to be ~ 39,500 g mol−1 (Additional file 1: Figure S2.3(f)).
Characterization of noncovalent graft copolymer micelles
Due to their amphiphilic nature, in the aqueous solution, PHEMA-g-(PCL-BM):(CD-star-PMAA-b-PNIPAM) molecules can self-assemble into micelles through host–guest interactions (Scheme 2). The hydrophobic PHEMA-g-(PCL-BM) section assembled as the core, while the hydrophilic CD-star-PMAA-b-PNIPAM section extended in the shell to afford the micelle solubility in water.
Figure 2A shows the DLS results of PHEMA-g-(PCL-BM: β-CD-star-PMAA-b-PNIPAM) micellar solutions at various weight ratios of BM moieties (guest) to β-CD moieties (host). It indicates that the average hydrodynamic diameters (Dh) of the micelles are 158, 162, 134, 182, and 167 nm and its relative average polydispersity index (PDI) are 0.255, 0.242, 0.210, 0.27, and 0.4 when guest: host weight ratios = 1:1, 1:2, 1:3, 1:4, and 2:1 respectively. The guest/host weight ratio in the range from 1:1 to 1:4 has a low effect on the micelle size and the size distribution. Similar results have been reported in the literature [39, 40]. Based on these results, the weight ratio of PHEMA-g-(PCL-BM) to (β-CD-star-(PMAA-b PNIPAM) in the micelles was fixed at 1:3 in the subsequent measurements, which had a suitable size and size distribution of micelles in water.
The self-assembly of PHEMA-g-(PCL-BM: CD-star-PMAA-b-PNIPAM) was characterized by measuring the CMC value of the blank micelles using the fluorescence spectroscopy method and by employing pyrene as a fluorescent probe at 25 °C. The fluorescence intensity ratio of I384/I373 versus log C is plotted in Fig. 2B. The I384/I373 ratio remained almost constant at low graft copolymer concentrations. When the copolymer concentration reached a specific value, the total fluorescence intensity ratio increased remarkably. This is a reflection of the graft copolymer micelles aggregation. The CMC value of PHEMA-g-(PCL-BM: CD-star-PMAA-b-PNIPAM) in an aqueous solution was 0.0177 mg mL−1 determined from Fig. 2B. The CMC is an effective parameter characterizing micellar stability, and a lower CMC value indicates relatively higher stability [41].
In this study, the BM moiety of the PHEMA-g-(PCL-BM) was fitted into the β-CD cavity of the CD-star-PMAA-b-PNIPAM via inclusion complexation, forming noncovalent graft copolymer micelles. In order to confirm the inclusion complexation between the host and guest units, the 2D-NOESY NMR spectrum of PHEMA-g-(PCL-BM):(CD-star-PMAA-b-PNIPAM) copolymer micelles was prepared. The cross-peaks in this spectrum identify the protons of guest and host molecules undergoing “through space” dipolar interactions and the formation of inclusion complexes [42,43,44]. As shown in Fig. 2C, the observed cross-peaks indicate the interaction between BM protons and the inner protons of β-CD in an inclusion complex. The signals appeared at δ 6.8–7.8 ppm [45] attributed to the BM moieties of guest copolymer demonstrate cross-peaks, resulting from dipolar interactions with the signals that occurred at 3.5–3.8 ppm [15, 42, 46] ascribed to the C(h)-H and C(f)-H protons (see Fig. 1 (C-3)) located inside the cavity of β-CD moieties in the host copolymer, which is in accordance with the Yang et al. report [47]. This phenomenon could support the formation of inclusion complexes via host–guest interaction between the BM moieties with β-CD cavities.
In order to demonstrate the successful association of BM units and CD units, the fluorescence of the complexes in the mixed solvent (DMSO/PBS, pH 7.4) was also studied. As shown in Fig. 2E, the PHEMA-g-(PCL-BM) concentration was fixed at 0.5 mg/ml, whereas the concentration of CD-star-PMAA-b-PNIPAM was increased from 0 to 1.6 mg/ml. The pure PHEMA-g-(PCL-BM) solution showed typical fluorescence at 378 nm. The increase in CD-star-PMAA-b-PNIPAM concentration from 0 to 1.2 mg/ml led to a marked decrease in the fluorescence intensity of BM moieties, because a less hydrophobic environment was formed by the association of BM moieties with CD moieties. Therefore, the decrease in the fluorescence intensity of BM moieties upon the addition of CD-star-PMAA-b-PNIPAM suggests the formation of an inclusion complex between the BM moieties and CD moieties of PHEMA-g-(PCL-BM) and CD-star-PMAA-b-PNIPAM. It is noteworthy that the further increase of the CD-star-PMAA-b-PNIPAM concentration from 1.2 to 1.6 mg/ml had no obvious influence on the fluorescence intensity of BM moieties. It could be reasonably explained that the BM moieties were almost fully associated with β-CD of CD-star-PMAA-b-PNIPAM, which similar results reported by some related works [48].
Further investigation into the successful association of BM units and CD units was conducted using FT-IR spectroscopy and AFM as complementary methods. FT-IR spectroscopy is an excellent analytical tool to confirm the formation of inclusion complexes via noncovalent interaction between host and guest molecules by identifying considerable changes in intensity, characteristic peak position, and shape [38, 49, 50]. FTIR spectra were firstly utilized to evidence the formation of graft copolymer micelles via host–guest interactions. The FTIR spectra of the PHEMA-g-(PCL-BM) (guest), CD-star-PMAA-b-PNIPAM (host), and PHEMA-g-(PCL-BM: CD-star-PMAA-b-PNIPAM) (micelle) are shown in Fig. 2D.
Noncovalent interactions between β-CD and BM in the PHEMA-g-(PCL-BM: CD-star-PMAA-b-PNIPAM) micelles causes to minimize the energy of included part of BM and reduce the peak intensity of relevant frequency.
The FTIR spectrum of the guest is characterized by absorption peaks at 1372 cm−1 (for C−N stretching vibration), 732 and 1190 cm−1 (for C−H vibration), 1244 cm−1 (for C−C stretching vibration) [35,36,37]. All of these peaks are induced by benzimidazole. In the spectrum of the micelle, the absorption bands at 732 and 1244 cm−1 disappeared, and the bands at 1041, 1190, and 1372 cm−1 aroused from PHEMA-g-(PCL-BM) reduced, which is due to the change in the environment of the guest molecule after inclusion in the cavity of CDs. In addition, in the guest spectrum, the bands at 1041 and 1726 cm−1 were the C−O−C and C=O vibrations from PHEMA-g-PCL, while these peaks decreased in the spectrum of the micelle. Guest and micelle spectra show intense bands at 3417−3480 cm−1 for the tertiary nitrogen of benzimidazole ring stretching vibrations. These bands in the guest spectrum and the broad band of host spectrum at 3442 cm−1 are merged in the inclusion complex. In the host spectrum, the characteristic peak at 948 cm−1 was assigned to the sugar ring skeleton from cyclodextrins in CD-star-PMAA-b-PNIPAM [51], which was also perceived in the spectrum of graft copolymer micelles. The characteristic stretching vibration of PNIPAM at wavelengths of 1560 and 1638 cm−1 remained. These results suggest that the hydrophobic BM moieties inserted into the hydrophobic cavity of β-CD moieties to prepare PHEMA-g-(PCL-BM: β-CD-star-PMAA-b-PNIPAM) micelles via host–guest interaction.
Nuclear magnetic resonance (1HNMR) spectroscopy ascertains the inclusion phenomena of the guest molecule inside the β-cyclodextrin cavity as the host molecule [46, 50, 52]. The noncovalent graft copolymer micelles formed in host–guest self-assembling systems were also investigated using the 1HNMR method, as shown in section S2.4, supplementary information.
AFM is a suitable non-invasive technique for observing the surface texture of deposited films and measuring the surface roughness at micro-/nano-scale [51, 53]. Both 2D and 3D AFM images of PHEMA-g-(PCL-BM) as guest and PHEMA-g-(PCL-BM: CD-star-PMAA-b-PNIPAM) inclusion complex are shown in Fig. 2. The AFM images display nodules on the top surface visible as bright high peaks, while the pores are seen as dark depressions. Visualization of surface topography of PHEMA-g-(PCL-BM) (Fig. 2F) and PHEMA-g-(PCL-BM: CD-star-PMAA-b-PNIPAM) inclusion complex (Fig. 2G) by AFM analysis revealed changes in surface morphologies when the including complexes formed between β-CD moieties and BM moieties. The change in the surface topography is proportional to the change in the pore size of the compound and provided strong evidence to confirm the formation of the PHEMA-g-(PCL-BM: CD-star-PMAA-b-PNIPAM) inclusion complex. The average roughness values for PHEMA-g-(PCL-BM) and PHEMA-g-(PCL-BM: CD-star-PMAA-b-PNIPAM) inclusion complexes were 9.06 nm and 5.61, respectively. This reveals that PHEMA-g-(PCL-BM) has a rough surface, and when BM molecules are embedded in the cavities of β-CD via host–guest interaction, the surface roughness of PHEMA-g-(PCL-BM) is changed into a smooth surface. Based on the results of the AFM studies, the inclusion complex obtained by combining the host and guest segments were indeed graft copolymers, indicating that polymerization at each step was successful.
Morphological characterization
The FESEM micrograph of blank graft copolymer micelles is presented in Fig. 3A. FESEM images of the particle morphology and size distribution of micelles indicate the assemblies are regular spherical structures with an average diameter of around 80 nm.
Furthermore, transmission electron microscopy (TEM) results confirmed the spherical micellar morphology and uniformity of the blank graft copolymer micelles. TEM image of the micelles formed by host–guest interaction with a concentration of 0.2 mg ml−1 at 25 ºC is shown in Fig. 3B. The average size observed by the TEM image (~ 80 nm) was in accordance with that measured by SEM (Fig. 3(A-1) and (B-1)).
Evaluation of size and zeta potential by (DLS) technique
The size and zeta-potential of the micelles are among crucial factors affecting their properties and performances in vivo. The size between 60 and 200 nm is the suitable size of nanocarriers for cancer therapy, and in this size range, is expected to restrict their uptake by the mononuclear phagocyte system and permit for passive targeting of cancerous or inflamed tissues through the enhanced permeation and retention (EPR) effect [54, 55]. The hydrodynamic diameter (Dh), polydispersity index (PDI), and zeta potential of the micelles were measured via DLS, as shown in Fig. 3C, D. It was found that the Dh of the micelles was about 134 nm at 25 °C and the PDI value was about 0.216, which shows a narrow size distribution. The present observation showed that the average size of micelles measured by DLS differed from those obtained from TEM and SEM analyses. This is mainly due to the micelles were swollen in water in the DLS analysis, and the hydrated diameter of micelles in the liquid is determined [45, 48]. As seen in Fig. 3D, the graft copolymer micelles revealed a high negative zeta potential value (− 23.6 mV) due to the presence of many carboxylate groups of the PMAA segment in polymer chains bonded onto the surface of micelles, which is in accordance with the Yang et al. report [56].
Thermo-response of the noncovalent graft copolymer micelles
The main aim of incorporating NIPAM units within these amphiphilic graft copolymers was to bring about a thermo-sensitive behavior to prepared micelles. The polymers containing PNIPAM indicate lower critical solution temperature (LCST) behavior, which corresponds to a thermo-sensitive hydrogen bonding ability of amide groups with the water solvent. LCST of PNIPAM is altered through the attachment of PMAA chains, and it is expected to be lowered if the PMAA block is hydrophobic (at low pH) and raised if the PMAA block is hydrophilic (at high pH) [57]. The temperature-responsive behavior of the graft copolymer micelles (with a host to guest ratio of 3 to 1 (w/w)) was investigated by turbidimetry and DLS measurements at various temperatures (from 30 to 50 °C).
As the first step, visual observations were carried out to study the temperature-responsive behavior of PHEMA-g-(PCL-BM: CD-star-PMAA-b-PNIPAM) graft copolymer micelles in water. The results obtained from visual observation are shown in Fig. 4a, b). These results showed that the micellar solution was transparent below 40 °C, while turbid above 40 °C. In the next step, turbidity variation of the samples at various temperatures was measured using a UV–vis spectrophotometer at a wavelength of 600 nm, and the results are shown in Fig. 4a. In this way, the LCST was determined from the transmittance of samples containing polymeric micelles as a function of temperature, as depicted in Fig. 4a. Below the LCST, the PNIPAM block was hydrophilic and water-soluble, owing to the formation of hydrogen bonds between the water and amide groups of PNIPAM, and the transmittance percentage was approximately 100%. When the temperature reached above the LCST, the hydrophobic interactions became dominant, and the transmittance reduced since the PNIPAM blocks became hydrophobic and started to aggregate in the aqueous solution. It is worth noting that the LCST value of the graft copolymer PHEMA-g-(PCL-BM: β-CD-star-PMAA-b-PNIPAM) micelles was pH-dependent. The incorporation of hydrophilic PMAA block into PNIPAM block increased the LCST of the copolymer and decreased the phase transition rate by stabilizing polymer dissolution [58]. In this work, the same phenomenon was observed.
On the other hand, incorporating hydrophobic guest moieties into the polar cavities of β-CD as host moieties prevents further increase of LCST [17]. As shown in Fig. 4a, the transmittance of the graft copolymer micelles reduced from 100% to almost 80% with the enhancement of temperatures from 31 to 50 °C in neutral pH, being in accordance with the previously reported studies [31, 57, 59]. The LCST of the graft copolymer PHEMA-g-(PCL-BM: β-CD-star-PMAA-b-PNIPAM) micelles was found to be about 40−410 °C.
Further investigation into the thermoresponsive behavior of micelles was conducted using DLS measurements as a complementary method. The results obtained by DLS at various temperatures are presented in Fig. 4b. When the temperature of the sample solution was reached its LCST value, the association of aggregates occurred, and the diameter of aggregates significantly increased from 134 to 405 nm as the temperature increased from 25 to 410 °C. A further increase in temperature caused a slight reduction in the diameter of the aggregates (450 °C), presumably because the interactions between hydrophobic groups became dominant and further heating the solutions triggered the shrinking of the aggregates as a result of dehydration [60, 61].
pH-Response of graft copolymer micelles
The environment of tumor tissues is different from that of the normal tissues in many aspects, including high intracellular adenosine triphosphate (ATP) concentration, a low pH value, and overexpression of some biological enzymes, which is the basis for the design of intelligent drug carriers [62, 63].
The pH-responsive noncovalent graft copolymers can be defined as polyelectrolytes that include in their backbone or side chains weak acidic (e.g., carboxylic and sulfonic acids) or basic groups (e.g., amines, imidazole, and pyridine) that either accept or donate H+ ions in response to environmental pH changes [64]. Our results showed that micelles formed from PHEMA-g-(PCL-BM: β-CD-star-PMAA-b-PNIPAM) have a pH-responsive behavior due to the presence of benzimidazole units and PMAA units on the graft copolymer chains.
Under acidic conditions, host–guest interactions between BM segments and β-CD segments can be cleaved due to the BM could be protonated in the acidic environment and causes dissociation of inclusion complexes [47, 65]. On the other hand, at lower pH values, carboxyl groups (−COOH) of the PMAA moieties are protonated, and hydrophobic interactions dominate, leading to reduction of the volume of the polymer containing the carboxyl groups. With increasing the pH, −COOH groups start to deprotonate, resulting in a high charge density in the polymer. Due to Coulomb repulsion, the negative charge of carboxylate ions would induce repulsive forces and increase the free volume [58]. The pH at which these changes occur is called “transition pH”, which depends on the pKa value of the polymer [66].
The pH-responsive behavior of the micelles from host–guest mixtures in deferent buffer solutions was investigated through UV–Vis transmittance at a wavelength of 600 nm and transmission electron microscopy (TEM). Figure 4c demonstrates transmittance changes of the polymeric micelles with changes in pH. It can be seen that the micelles displayed a significant increase in transmittance at ca. pH 5.5, which was very near to the pKa of PMAA units (about 5.6 [67, 68]). At pH below the pKa value (5.5), PMAA exhibits a marked pH-induced conformational transition, and owing to the formation of aggregates of the hydrophobic segments after the disassociation of the inclusion complexes, the transmittance had relatively lower values, being in accordance with the previously reported studies [68, 69].
The effect of pH on the size of the graft copolymer micelles was investigated through TEM. Based on TEM analysis, it was found that the blank graft copolymer micelles had a nanosized spherical shape with a size of approximately 80 nm at pH 7.4, while the particle size demonstrated a significant increase at pH 4.5, as shown in Fig. 4d.
Loading of DOX in noncovalent graft copolymer micelles
Generally, there are three primary methods for loading drugs into polymer micelle cores, (1) physical entrapment or solubilization, (2) polyionic complexation, and (3) chemical conjugation [25]. Although the covalent conjugation strategies have some limitations for loading the drugs due to the chemical modification of drugs’ structure, which may cause drugs to be less effective, the physical encapsulation via hydrophobic interactions or charge does not lead to any change in the chemical structure of the drug [70]. The electrostatic interactions between the drug and the polymeric matrix are one of the most important mechanisms in the physical encapsulation method. Additionally, hydrogen bonding could also act as the main force between drugs and carriers [70, 71]. DOX is a potent chemotherapeutic antineoplastic agent, but it has a short in vivo biological half-life and seriously injured the healthy cells when killing the cancerous cells [72]. In this research, to verify the feasibility of using the self-assembled micelles based on graft copolymer as a 'smart' drug carrier in cancer treatment, DOX.HCl was selected as a model hydrophilic drug and loaded into the fabricated micelles by a dialysis method [33, 48, 45]. Although DOX.HCl displays water solubility character, it keeps the identical physicochemical and biological properties as the hydrophobic form of DOX, while it still has a main hydrophobic structure [70].
The Dh, polydispersity index, and zeta potential of DOX-loaded micelles were measured through DLS, as summarized in Fig. 5a. The particle size of DOX-loaded micelles indicated that encapsulation of DOX into micelles increased the diameters of micelles, which similar results reported by some related works [73, 74]. The pKa of DOX is 8.3, so it has positively charged at pH ≤ pKa due to the existence of the amino groups. The amine functional groups (−NH2) in the DOX chemical structure can absorb the protons and convert them to the cationic form (−NH3+) [70]. Thus, it was attracted to the negatively charged micelles and effectively loaded into the core of the micelles based on the hydrophobic and electrostatic interactions between the drug molecules and the polymer chains [70, 75]. Furthermore, DOX could also be entrapped into polymeric micelles architecture through hydrogen bonding between the side chains of graft copolymer and DOX. Therefore, after DOX loading on micelles, owing to the presence of electrostatic interaction between carboxylate groups of the PMAA segment in polymer chains bonded onto the surface of micelles and DOX with positive charge [56, 76], the zeta potential of the drug-loaded micelles slightly reduced from − 23 to − 19 mV. The LC and EE percentages of DOX in the graft copolymer micelles (polymer to drug ratio of 10 to 1) were 9.73% and 97.3%, respectively. These high loading and encapsulation efficiency may be related to the hydrophobic PCL blocks in micelles core and the hydrophobic cavities of β-CD, which enable them to load a large amount of drugs [45, 75]. Therefore, the assembled micelles based on the graft copolymer (PHEMA-g-(PCL-BM: β-CD-star-PMAA-b-PNIPAM) can suggest promising features for their use as controlled drugs carriers.
Release of DOX from noncovalent graft copolymer micelles
The major subject for designing an effective drug carrier is that it can maintain its stability under normal physiological conditions (pH ~ 7.4) but selectively release drug molecules by sensing pH decrease in the vicinity of cancer tissue (pH ≤ 5) and/or the endosome-lysosome (pH ~ 4.5) [77]. To evaluate the process of DOX release behavior of the fabricated PHEMA-g-(PCL-BM: β-CD-star-PMAA-b-PNIPAM) micelles, two temperature values (37 and 42 °C) and three different pH values (7.4, 5.7, and 4.5), were chosen to emulate the normal physiological and cancerous tissue conditions. As seen in Fig. 5(c-1) and (c-2), under the physiological conditions (pH 7.4), the DOX-releasing efficiencies remained relatively stable, and only about 32 and 35% of the DOX were released within 24 h at 37 and 42 °C, respectively. In contrast, under cancerous conditions (pH 5.7 and 4.5), the DOX release was significantly accelerated, especially at pH 4.5. At a pH value of 4.5, the BM molecules were protonated, which led to the dissociation of host–guest inclusion complexes between BM segments and cyclodextrin segments [17, 48, 69]; approximately 70 and 85% of the loaded DOX were released from fabricated micelles in the first 24 h at 37 and 42 °C, respectively.
On the other hand, the fast drug release at a temperature above the LCST of micelles was due to the that the collapse of PNIPAM segments located in the shell increased the spatial distance between polymer chains and shorten release way [41]. The present observation showed that sustained release is carried out by the departure of cyclodextrins and contraction of temperature-sensitive segments, as shown in Fig. 5(b-1) and (b-2).
All results proved that the designed nanocarrier based on the host–guest inclusion complex was dual-responsive and should be suitable for cancerous drug delivery systems.
In vitro biocompatibility evaluation and cytotoxicity assay
The biocompatibility of the prepared micelles, as well as the cytotoxicity of DOX-loaded micelles, were investigated by the colorimetric MTT assay against MCF-7 cells in the time period of 48 h, and the obtained results were compared with the cytotoxicity effect of free DOX as the reference, as shown in Fig. 6. It was observed that the graft copolymer micelles alone had much low cytotoxicity on MCF-7 cell lines (Fig. 6a). The determined half-maximal inhibitory concentration (IC50) values of free DOX and DOX-loaded polymeric micelles against MCF-7 cells were ca. 18.29, and 1.75 μg/ml, respectively. As can be seen in Fig. 6b, compared with blank polymeric micelles, the viability of the cells treated with DOX-loaded polymeric micelles at a very low dose of drug exhibited a rapid decrease. The cell viability rate of DOX-loaded polymeric micelles at the drug dose of 50 μg/ml was almost 12.7%, while the cell viability rate of free DOX at the same drug dose was only 23.1%. This observation displayed that the cell growth inhibition of DOX-loaded polymeric micelles was higher than that of free DOX at the same drug dose. The higher tumor growth inhibition efficacy observed for the DOX-loaded polymeric micelles can be attributed to the improved tumor accumulation of DOX in the MCF-7 cells and fast intracellular drug release [45]. The cells treated with blank micelles compared with untreated cells demonstrated no significant morphological changes and revealed their evenly and intact shapes.
We further investigated the viability and morphological changes of the MCF-7 cells after treatment with various formulations for 48 h, as shown in Fig. 6c. MCF‑7 breast cancer cells have a shuttle, triangle, or irregular shape, and they have a habit of clumping into large aggregates [78]. Under microscopic observation, compared with the control group, the tumor cells in blank micelles-treated groups had almost no change, but DOX treatment destroyed the compact structure and shuttle morphology of tumor cells. These results were entirely consistent with the viability of the MCF-7 cells after treatment with different doses of the drug-free micelles. In contrast, in the DOX-loaded micelles-treated group, the structure of tumor cells was severely disrupted and many cells separated from tumor cells, suggesting that the drug was endowed with strong infiltration capability and effective antitumor depending on the micelle delivery system. These results implied that DOX was efficiently delivered into MCF-7 cells by PHEMA-g- (PCL-BM: β-CD-star-PMAA-b-PNIPAM) micelles, which corroborates with the MTT results in this study.
Morphological assessment of apoptotic cells using DAPI staining
The 4,6-diamidino-2-phenylindole (DAPI) is a cell-permeable fluorescent compound that binds to fragmentation of DNA and condensation of chromatin as changes in the nucleus [76]. In this regard, DAPI staining was utilized as a complementary assay to assess the morphological alterations induced by apoptosis in MCF-7 cells. MCF-7 cells were treated with free DOX and drug-loaded PHEMA-g- (PCL-BM: β-CD-star-PMAA-b-PNIPAM) micelles with two concentrations (in the range of IC50 values and drug dose of 50 μg/ml). The morphological changes in cell nuclei after 48 h treatment were observed by fluorescence microscopy, and the results are shown in Fig. 7. The images of cells treated with free DOX and DOX-loaded PHEMA-g- (PCL-BM: β-CD-star-PMAA-b-PNIPAM) micelles demonstrated the signs of apoptosis such as cell shrinkage, nuclear fragmentation, loss of cell–cell contact, and chromatin condensation. In comparing these groups, the MCF-7 cells treated with DOX-loaded micelles revealed high-level apoptosis evidence in contrast to the free DOX, but no apoptotic cells were detected in the control group.