A novel ‘smart’ PNIPAM-based copolymer for breast cancer targeted therapy: Synthesis, and characterization of dual pH/ temperature-responsive lactoferrin-targeted PNIPAM-co-AA

Osama R.M. Metawea a,b, Mona A. Abdelmoneem c,d, Nesreen Saied Haiba e, Hosam H. Khalil a, Mohamed Teleb d,f, Ahmed O. Elzoghby d,g,*, Asmaa F. Khafaga h, Ahmed E. Noreldin i, Fernando Albericio j,k,l, Sherine N. Khattab a,d,**

Abstract

Despite the active research towards introducing novel anticancer agents, the long-term sequelae and side effects of chemotherapy remain the major obstacle to achieving clinical success. Recent cancer research is now utilizing the medicinal chemistry toolbox to tailor novel ‘smart’ carrier systems that can reduce the major limitations of chemotherapy ranging from non-specificity and ubiquitous biodistribution to systemic toxicity. In this aspect, various stimuli-responsive polymers have gained considerable interest due to their intrinsic tumor targeting properties. Among these polymers, poly(N-isopropylacrylamide (PNIPAM) has been chemically modified to tune its thermoresponsivity or even copolymerized to endow new stimulus responsiveness for enhancing tumor targeting. Herein, we set our design rationale to impart additional active targeting entity to pH/temperature- responsive PNIPAM-based polymer for more efficient controlled payloads accumulation at the tumor through cellular internalization via synthesizing novel “super intelligent” lactoferrin conjugated PNIPAM-acrylic acid (LF- PNIPAM-co-AA) copolymer. The synthesized copolymer was physicochemically characterized and evaluated as a smart nanocarrier for targeting breast cancer. In this regard, Honokiol (HK) was utilized as a model anticancer drug and encapsulated in the nanoparticles to overcome its lipophilic nature and allow its parenteral administration, for achieving sustainable drug release with targeting action. Results showed that the developed HK- loaded LF-PNIPAM-co-AA nanohydrogels displayed high drug loading capacity reaching to 18.65 wt.% with excellent physical and serum stability. Moreover, the prepared HK-loaded nanohydrogels exhibited efficient in vitro and in vivo antitumor activities. In vivo, HK-loaded nanohydrogels demonstrated suppression of VEGF-1 and Ki-67 expression levels, besides inducing apoptosis through upregulating the expression level of active caspase-3 in breast cancer-bearing mice. Overall, the developed nanohydrogels (NGs) with pH and temperature responsivity provide a promising nanocarrier for anticancer treatment.

Keywords:
PNIPAM
Lactoferrin
Honokiol
Smart polymers Breast cancer

1. Introduction

Breast cancer is the most common cancer among women and the second cause of cancer death globally according to GLOBOCAN 2018 [1]. Many reports forecasted more expected breast cancer cases by 2030 [2,3]. In recognition of the global burden posed by breast cancer, extensive efforts are actively ongoing to introduce novel chemotherapeutics to clinical application. However, the conventional anticancer therapy remain a major clinical concern. Modern cancer research is now utilizing new strategies of the medicinal chemistry toolbox to tailor novel ‘smart’ carrier systems that can reduce the major limitations of chemotherapy ranging from non-specificity and ubiquitous biodistribution to systemic toxicity [4]. In this aspect, stimuli-responsive polymers have gained considerable interest in cancer therapy [5]. These polymers can undergo sharp changes in properties, e.g. solubility, conformation, hydrophilic/hydrophobic balance or payload release in response to endogenous (pH, redox or hypoxia) or exogenous stimuli (ultrasound or temperature). These changes are usually reversible; where the system returns to its initial state after removing the triggering stimulus. Based on differences between normal and cancerous tissues, this behavior can be successfully utilized to achieve controlled drug release at the target sites with minimal dose alteration and toxicities consequently enhancing therapeutic efficacy and minimizing side effects. Various polymers have been synthesized and explored for preparing stimuli-responsive anticancer delivery systems [6–8].
To date, poly(N-isopropylacrylamide) (PNIPAM) is among the most substantially investigated temperature-responsive biomaterials [9,10]. It is a biocompatible polymer and thermoresponsive with a lower critical solution temperature (LCST) at 32 ◦C. When PNIPAM is heated beyond its LCST, it can undergo reversible coil-to-globule conformational changes [11–13]. This thermal responsivity allowed sequestering and releasing therapeutic cargo in tumor site [14–16], as a “smart nanocarrier”. Several studies have shown that PNIPAM can be chain-end functionalized with various functional groups or copolymerized for tuning its thermal responsiveness (LCST) to the desired temperature range [17,18] or even imparting pH sensitivity [17,19]. A prime example of these modifications is copolymerization of N-isopropylacrylamide (NIPAM) and acrylic acid (AA) to afford PNIPAM-co-AA with combined thermo- and pH-responsive entities [19, 20]. Hence, several studies demonstrated various synthetic protocols to modulate the PNIPAM-co-AA properties [17,19,21–23].
Besides tailoring such multi-responsive polymers, conjugation with active targeting ligands can also enable more efficient controlled payloads release at the tumor [24–26]. Lactoferrin (LF), a natural cationic iron-binding glycoprotein [27,28], has been successfully utilized as ligand for breast cancer targeting [29], where it can bind efficiently to many receptors as low-density lipoprotein (LDL) and lactoferrin receptors (LRP1, LRP2) that are over expressed in breast cancer cells [30–32]. Interestingly, LF itself has shown promising anticancer activity against breast cancer [33]. Mechanistic studies reported that such activity relies on its ability to induce apoptosis and reduce cancer cell invasion [33–36]. Beside its anticancer potential, LF showed efficient anti-inflammatory [37], antioxidant, cytoprotective [38], and antimicrobial activities [39]. Moreover, LF has been approved as a safe ingredient for various applications by the European Food Safety Authority. Thus, it has been included in several injectable nanoformulations without eliciting immunogenic reactions [40,41].
With the aforementioned advances in designing smart targeting systems, cancer research has been directed to utilize natural or natural- derived anticancer agents [42]. Various phytochemicals have already showed excellent potential in breast cancer prevention and therapy. Also, more ones are frequently examined in search for new bioactive molecules [43]. Recently, honokiol (HK); a bi-phenolic lignin isolated from Magnolia officinalis, a widely distributed Asian plant [44] has attracted considerable interest due to its efficient chemopreventive and anticancer properties ranging from inhibiting proliferation and angiogenesis to suppressing metastasis and reversing resistance [45]. Besides, HK was reported to be safe in animal models, either alone or as part of magnolia extract [46]. Mechanistic studies showed that HK targets multiple signaling pathways with great significance during tumor initiation and progression such as nuclear factor-κB (NF-κB) [47], mitogen-activated protein kinases (MAPK) [48], mammalian target of rapamycin (m-TOR), epidermal growth factor receptor (EGFR) [48], signal transducers [49] and others. In breast cancer, HK inhibits cell invasion and metastasis through increasing the expression of tumor suppressor LKB1 which led to activation of AMPK thus knocking down the mTOR signaling [50]. In addition, HK inhibits breast cancer cell migration by targeting nitric oxide and cyclooxygenase-2 [51], and sensitizes the tumor cells for therapeutics by downregulating P-glycoprotein [51]. Furthermore, HK was found to induce antiangiogenic activity via downregulation vascular endothelial growth factor (VEGF) [52]. Also, HK exerts its antitumor activity through inducing apoptosis via upregulating caspase-3 expression level. All these findings point to HK as potential phytotherapy for breast cancer. Though, HK’s poor aqueous solubility has hampered its clinical application. In attempt to overcome this issue, honokiol encapsulation has been assessed [53,54].
Herein, we report a novel targeted stimuli-responsive LF-PNIPAM- co-AA NGs for delivery of HK in breast cancer therapy. First, PNIPAM- co-AA copolymer was synthesized as dual stimuli-responsive polymer by graft copolymerization and decorated by LF via carbodiimide coupling as active targeting approach to maximize accumulation and specific drug release in breast cancer cells. The successful synthesis PNIPAM-co- AA was confirmed by IR, H1-NMR and MALDI TOF-MS. Second, HK was then loaded into the NGs core to overcome its hydrophobicity, allowing its parenteral administration and achieving sustainable drug release. The developed NGs were assessed in vitro and in vivo to compare the antitumor activity of HK loaded LF-PNIPAM-co-AA NGs with the free HK.

2. Results and discussion

2.1. Synthesis and characterization of PNIPAM-co-AA copolymer (PNIPAM-co-AA(1:1), P1 & PNIPAM-co-AA(4:1), P2)

PNIPAM-co-AA copolymers P1 & P2 were successfully synthesized by graft copolymerization of NIPAM and different amounts of AA using the cross-linking agent N,N-methylene bisacrylamide (BIS), the phase transfer agent sodium dodecyl sulfate (SDS) and potassium persulfate (KPS) as initiator. The free radical polymerization was initiated by KPS under nitrogen, by heating at 70 ± 2 ◦C for 4 h. The initiator KPS underwent thermal homolytic dissociation and the formed free radicals reacted with monomers then underwent a series of chain growing reactions (chain propagation) in presence of both BIS and SDS to produce NGs of PNIPAM-co-AA copolymer (Scheme 1) [55,56]. Termination occurred by recombination and disproportionation reactions. The dialyzed solution of nanohydrogel was lyophilized for further characterization.
It was found that the synthesized copolymer P1 with high ratio of AA displayed much higher particle size 645.5 nm with more negative zeta potential -35.6 mV compared to PNIPAM-co-AA copolymer (P2) with lower amount of AA which exhibited average particle size 126.7 nm, PDI 0.375 with zeta-potential -28.6 m (Table 1). In fact, acrylic acid has higher hydrophilicity compared to NIPAM. In addition, it forms the shell of NPs that embrace the PNIPAM core, through hydrogen bonding with the aqueous solutions. Therefore, the particle size of the copolymer increased with increasing the AA amount [57].
The FTIR study of the prepared PNIPAM-co-AA copolymer showed absorption band at wave number 3434 cm− 1 corresponding to the NIPAM monomer NH stretching (Fig. 1A). A broad band at 3600− 2500 cm− 1 assigned to the OH group of acrylic acid is also observed [55]. The FTIR spectrum of PNIPAM-co-AA(1:1) P1 displayed a strong sharp absorption band at 1717 cm− 1 which corresponding to the carboxylic carbonyl groups of AA monomer that is incorporated during the copolymerization. The intensity of this band is decreased in the spectrum of PNIPAM-co-AA(4:1) P2 which could be correlated to the incorporation of less amount of AA in the copolymerization of P2. Another strong sharp peak at 1644 cm− 1 is observed in P2 spectrum which corresponding to the amidic C––O groups of NIPAM (Fig. 1A). While in P1 spectrum, this peak was noticed with lower intensity due to the reduction of the NIPAM amount incorporated during its polymerization.
The 1H NMR spectrum of P2 copolymer in DMSO-d6/D2O (Fig. S1) shows two singlet peaks at chemical shift 1.14 and 1.281 ppm assigned to the two CH3 groups of NIPAM. Besides, 4 multiplets are observed at chemical shift ranges 1.50–1.80, 2.00–2.50, 3.60–3.70, 3.80–4.10 ppm corresponding to the (C–H and CH2) protons of P2. The molecular weight range of the prepared copolymers was measured using MALDI TOF-MS analysis. PNIPAM-co-AA copolymers P1 and P2 showed a molecular weight range of 1100–5500 and 2100− 7300 Da (Fig. S2A, B), respectively.

2.2. Synthesis and characterization of LF-PNIPAM-co-AA conjugate (P3)

For active breast cancer targeting, LF has been used as a targeting ligand due to its high binding affinity to the overexpressed LF receptors on the breast cancer cells surface. Therefore, LF was then conjugated to PNIPAM-co-AA(4:1) P2 via carbodiimide coupling reaction. The carboxylic groups of PNIPAM-co-AA(4:1) P2 were activated by EDC.HCl/K salt of oxyma cocktail to the corresponding active esters which are more susceptible to nucleophilic substitution by LF amino groups [58–63]. Although lactoferrin contains hydroxyl groups which can act as handles for covalent bonds, it has already been verified that the amino groups in lactoferrin are more prone to this reaction due to their higher nucleophilicity. The FT-IR spectrum of LF-PNIPAM-co-AA(4:1) P3 copolymer confirms the successful conjugation of LF to PNIPAM-co-AA copolymer where the most characteristic bands of lactoferrin at 1643 cm− 1 (amide I) and 1467 cm− 1 (amide II) were observed, which provides evidence for the lactoferrin conjugation (Fig. 1B). The 1H NMR spectra of LF and LF-PNIPAM-co-AA(4:1) P3 copolymer (Fig. 1C, D) revealed that the PNIPAM-co-AA diagnostic peaks were masked by LF signals. The LF-PNIPAM-co-AA(4:1) P3 copolymer spectrum showed two very broad multiplet peaks at the range 0.50–1.80 and 1.81–2.40 ppm with larger integration in comparison with LF, that confirms the conjugation of LF and PNIPAM-co-AA(4:1) P2. Accordingly, MALDI-TOF-MS was carried out to confirm LF conjugation (Fig. S3A, B). It was found that one molecule of LF (molecular weight =82285.5 Da, Fig. S3A) was attached to each molecule of PNIPAM-co-AA(4:1) P2 (molecular weight range 2100− 7300 Da, Fig. S2B) resulting in LF-PNIPAM-co-AA(4:1) P3 copolymer (molecular weight 84000− 90000 Da, Fig. S3B).

2.3. LCST analysis of synthesized nanohydrogels

The phase transitions of PNIPAM-co-AA(4:1) P2 and LF-PNIPAM-co- AA(4:1) P3 NGs in aqueous solution were investigated by measuring the optical transmittance measurement along temperature range 25− 50 ◦C. As shown in Fig. 2A, both PNIPAM-co-AA(4:1) P2 and LF-PNIPAM-co- AA(4:1) P3 solutions has shown sharp decline in transmittance with the temperature increase. LCST of PNIPAM-co-AA(4:1) P2 was observed at ~34 ◦C. Below this temperature, the system was completely soluble in the water and above 34 ◦C the turbidity of the particles increased which could be correlated to the thermal breakage of hydrogen bonds between the water molecules and the polymer chains. The phase transition of LF- PNIPAM-co-AA(4:1) P3 occurred at a higher temperature (38 ◦C) than PNIPAM-co-AA(4:1) P2. The larger LCST was attributed to the incorporation of the hydrophilic lactoferrin in the polymer chains, where the phase transition of PNIPAM is a sort of hydrophilic–hydrophobic transition. Similar findings were observed in previous study where PNIPAAm-grafted gelatin, which also showed higher LCST (38 ◦C) compared with PNIPAM (32 ◦C), due to the conjugation of gelatin to PNIPAM [64].
The thermosensitive property of LF-PNIPAM-co-AA(4:1) P3 NGs may be also evaluated by the Zetasizer Nano-ZS90 via recording the change in particle size with temperature (Fig. 2B). When the temperature is increased from 25 to 50 ◦C, the particle size of LF-PNIPAM-co-AA(4:1) P3 NGs was decreased from 176.3–112 nm, because of the volume phase transition. This result is in agreement with previous study which has reported that the particle size of poly(n-isopropylacrylamide-co-((2- dimethylamino) ethyl methacrylate))-based nanoparticles was collapsed along with raising the temperature from 25 to 50 ◦C [65].

2.4. Preparation and physicochemical characterization of HK-loaded LF-

PNIPAM-co-AA nanohydrogels (HK/LF-PNIPAM-AA(4:1), P4) AA(4:1) P3 NGs hydrophobic core by simple solvent evaporation method [66]. The HK-entrapment efficiency (EE%) was up to 77.7 % with high drug-loading capacity 18.65 wt%, as determined by HPLC (Table 1). After HK entrapment into the LF-PNIPAM-co-AA NGs core, the average particle size slightly decreased from 176.3 (blank NPs) to 116.5 nm (Table 1, Fig. S4A).The particle size reduction indicates the formation of more compact hydrophobic cores due to increased hydrophobic interaction upon HK encapsulation [67]. It is worthy of mention that LF-PNIPAM-co-AA(4:1) P3 NGs zeta potential was decreased from -19.8 mV to -31.1 mV (Fig. S4B) after HK loading. This could be advantageous for maintaining good colloidal stability and serum stability of nanoparticles. Table 1

2.5. Solid state characterization

The FTIR spectrum of HK loaded LF-PNIPAM-co-AA(4:1) P4 NGs showed that HK characteristic peaks at 1644 cm− 1, 1498 cm− 1 corresponding to alkene and aromatic C––C stretching vibration respectively, which are overlapped with the absorption bands of LF (amide I, II). Besides, the HK peak at 3304 cm− 1 related to OH stretching was masked by the absorption peaks PNIPAM-co-AA(4:1) (Fig. 1B). The DSC thermogram of HK showed characteristic endothermic peak at 87.3 ◦C corresponding to HK melting temperature [68] (Fig. 2C). LF-PNIPAM-AA (P3) thermogram displayed endothermic peaks at 92 ◦C which is correlated to the presence of LF in polymer chains [69]. In the HK loaded LF-PNIPAM-co-AA (P4) thermogram there was obvious shifting to the LF peak to 122 ◦C, while the HK endothermic peak disappeared, which confirms the encapsulation of HK in an amorphous form. Wide angle XRD was performed for LF, PNIPAM-co-AA(4:1) P2, LF-PNIPAM-co-AA(4:1) P3 and HK loaded LF-PNIPAM-co-AA(4:1) P4 (Fig. 2D). The X-ray powder diffractogram of PNIPAM-co-AA(4:1) P2 nanohydrogel shows the disappearance of the defined reflections observed for NIPAM at 2θ = 15◦, 21◦, 29◦ (Fig. S5), and the appearance of a broad diffraction hump visible between 2θ = 12 and 24◦ [70], which indicates that P2 is semicrystalline, i.e. amorphous-crystalline. The diffractograms of lactoferrin contains two broad peaks at a diffraction angle around 10◦ and 21◦. While that of LF-PNIPAM-co-AA(4:1) P3 shows defined reflections at 2θ = 28◦, 41◦, 50◦, 67◦, in addition to a broad peak at a diffraction angle around 21◦, confirming the two polymers conjugation. Besides, the diffraction pattern of HK loaded LF-PNIPAM-AA P4 shows defined reflections at 2θ = 28◦, 41◦, 50◦, 67◦ similar to the one observed for P3, in addition to a larger broad peak, which is shifted to a diffraction angle around 24◦.

2.6. Morphological analysis, redispersibility test and physical stability

LF-PNIPAM-co-AA(4:1) P3 and HK loaded LF-PNIPAM-co-AA(4:1) P4 NGs were observed in the TEM micrographs as spherical-shaped particles in the nano-size range without any aggregations, attributed to the relative high stability of our nanoparticles (Fig. 2E, F). The size range was 103.44-110.85 nm, which is relatively smaller in size than that measured by DLS (116.5 nm), this could be due to the shrinkage induced by the dehydration of nanoparticles during preparation for TEM analysis. Besides, Fig. 3A shows SEM micrographs of HK loaded LF-PNIPAM- co-AA(4:1) P4 NGs. Physicochemical characterization of P1, P2, P3 and P4: particle size (PS), zeta potential, entrapment efficiency (EE), and drug content (DL) (n = 3).
After three months storage at 4 ◦C, the physical stability of HK loaded LF-PNIPAM-co-AA NGs P4 was evaluated (Fig. 3B, C). The HK loaded LF-PNIPAM-co-AA(4:1) NGs P4 showed after three months particle size of 145.8 nm, in comparison to the initially stored copolymer (116.5 nm). No dramatic changes were detected in zeta potential values after three months storage reflecting the stability of HK loaded LF-PNIPAM-co- AA(4:1) NGs. Lyophilization enhances the physical stability of nanoparticles and prevent release of therapeutics from NPs. Herein, the reconstituted lyophilized HK loaded LF-PNIPAM-co-AA(4:1) P4 demonstrated particle size of 123 nm with redispersibility index value of 1.036, where the acceptable range for Sf/Si ratio is 0.95–1.07 [71]. No significant changes are observed in particle size, PDI and zeta potential values after lyophilization (Table 1S). This result is in accordance with previous study which has reported that PNIPM can tolerate the lyophilization without using cryoprotectant [72].

2.7. In vitro drug release

In vitro release of free HK and HK loaded LF-PNIPAM-co-AA(4:1) P4 NGs at various temperatures and pH were carried out using dialysis bag method. As observed in Fig. 3D, free HK was almost completely released (90 %) after 8 h. HK release from the NGs at pH 7.4 and 37 ◦C exhibited biphasic pattern, where it showed burst release of about 42.1 % during the first eight hours followed by sustained release, reaching 54.1 % after 48 h. In neutral pH (7.4), we found that there is no significant effect to the temperature on the HK release. These findings are in accordance with previous study of Yar et al., who reported that the temperature has minimal influence on doxorubicin release from SPION-PNIPAM nanoparticles in neutral condition [73]. Nevertheless, a remarkable temperature-dependent release variation was detected at pH 5.5 which indicate that both acidic pH and temperature augment the HK release from LF-PNIPAM-co-AA(4:1) P4 NGs. Under acidic condition (pH 5.5), higher amount of HK, about 71.7 %, was released from NGs at 40 ◦C after 8 h compared with ~ 52.4 % HK release at 37 ◦C after 8 h. This result was attributed to the expelling of the encapsulated HK from the collapsed hydrogels at a temperature above its LCST (38 ◦C). In addition to, the enhanced pH sensitivity due to the presence of pH-responsive acrylic acid [74]. We can conclude that LF-PNIPAM-co-AA(4:1) NGs P4 are dual-stimuli responsive which would accelerate the drug release at tumor site through exploiting the acidic and hyperthermic tumor microenvironment.

2.8. Serum stability and hemocompatibility evaluation

Nanoparticles stability in serum is essential for parenteral administration. Unstable nanoparticles adsorb the serum proteins forming aggregates when incubated with serum [61]. In the current study, the HK loaded LF-PNIPAM-AA(4:1) NGs P4 was incubated with 10 % FBS to evaluate their serum stability (Fig. 3E). After adding the 10 % fetal bovine serum (FBS) solution (time 0), the HK-loaded LF-PNIPAM-co-AA(4:1) NGs P4 showed changes in particle size in comparison with the initially prepared NGs (from 123.0 nm to 162.2 nm). The PDI of both nanohydrogels ranged from 0.122 to 0.184 during the test period indicating high stability. After four hour incubation with fetal bovine serum, the particle size increased to 168.4 nm then decreased to 162.2 nm after six hours. This may be due to association and dissociation of proteins on the nanoparticles surfaces during incubation [75]. This obvious high serum stability may be attributed to the repulsive forces that could occur between the negative charge of prepared nanoparticles and the negatively charged serum proteins, which are superior to positively charged nanocarriers in clinical applications. Hemocompatibility is a major challenge in developing systems for parenteral delivery. Basically, nanoparticulate systems should not cause hemolytic events when administered intravenously. In the current study, in vitro hemolysis of the HK loaded targeted LF-PNIPAM-co-AA(4:1) (P4) was evaluated at two concentrations (0.25 and 0.5 mg/mL). Hemoglobin leakage from RBCs was utilized to quantitatively measure the membrane-damaging properties of nanohydrogels (Fig. 3F). At concentrations of 0.25 and 0.5 mg/mL, the prepared HK loaded LF-NIPAM-co-AA(4:1) NGs demonstrated 1.28 % and 1.6 % hemolysis which is lower than the accepted non-toxic level (5%) as indicated by the American Society for Testing and Materials (ASTMF 756− 00, 2000). These findings may be attributed to the negative charge on the nanohydrogels’ surface that renders it compatible with RBCs. Accordingly, the study demonstrated the safety of the prepared NGs for IV administration and the importance of utilizing LF in the fabrication of drug delivery carriers as being biocompatible, biodegradable and doesn’t cause red blood cell lysis.

2.9. In vitro cytotoxicity

The potential antitumor activity of free HK was compared to HK- loaded LF-PNIPAM-co-AA(4:1) NGs P4 against MCF-7 and A549 cancer cells via utilizing MTT assay at 24 h (Fig. 4A, B). The IC50 of HK-loaded LF-PNIPAM-co-AA(4:1) NGs P4 was 1.71-fold lower than free HK in A549 lung cancer cells (Fig. 4A,C). While in MCF7 cells the IC50 of HK-loaded LF-PNIPAM-AA nanoparticles was decreased by 2.7-fold in comparison to the free HK (4.23 ± 0.22 μg/mL) (Fig. 4C). MTT assay results in both cancer cell lines confirmed the enhancement of antitumor cytotoxicity of HK-loaded LF-PNIPAM-co-AA(4:1) NGs P4 compared to the free HK, accordingly, allowing its therapeutic dose reduction. This may be due to the capability of LF to efficiently uptake and accumulate the drug loaded nanoparticles in cancer cells. Moreover, the blank nanohydrogels P3 demonstrated negligible toxicity to MCF-7 cells for 24 h as evidenced by cell viability exceeding 94 %. This observation highlights their acceptable safety profile and negates any possible cytotoxicity of the copolymer.

2.10. In vitro cellular uptake and flow cytometry

In vitro cellular internalization of fluorescently labelled LF targeted- PNIPAM-co-AA(4:1) NGs P4 into breast cancer cell line (MCF-7) was evaluated after 4 and 24 h incubation by confocal laser scanning microscopy (CLSM) (Fig. 4D). The fluorescent dye rhodamine B isothiocyanate (RBITC) was conjugated to LF amino groups via its thiocyanate group to afford fluorescently labelled LF-PNIPAM-co-AA(4:1) NGs P4. Confocal microscopy showed bright red fluorescence in MCF-7 cells cytoplasm revealing that RBITC-labelled NGs successfully internalized in the cancer cells. The intensity of intracellular fluorescence was augmented with increasing incubation time in all samples, illustrating that the cellular internalization of RBITC- labelled NGs was time- dependent. This may be correlated to receptor-mediated endocytosis through LF moieties due to the overexpression of LF receptors on surface of MCF-7 cells [29,31,41,54]. The results were in accordance with the higher cytotoxicity revealed by LF-targeted NGs.  

3. In vivo anti-tumor efficacy

3.1. Tumor growth

The in vivo anti-tumor efficacy of HK/LF-PNIPAM-co-AA(4:1) NGs P4 was evaluated in Ehrlich ascites tumor (EAT)-bearing mice in comparison with free HK therapy. The treatment of EAT-bearing groups was maintained for three successive weeks during which tumors sizes were monitored. After treatment, the positive control group displayed the highest % increase in the tumor volume reaching 320 %. This was greater than those monitored in free HK (167 %) and HK-loaded LF- PNIPAM-co-AA(4:1) NGs P4-treated groups (138 %) (Fig. 5A). The tumor growth suppression of LF-targeted NGs could be attributed to the enhanced cellular uptake and accumulation in tumor tissue via lactoferrin receptor-mediated endocytosis. Obviously, HK-loaded LF-PNIPAM-co-AA NGs(4:1) NGs P4 achieved the most promising anticancer activity as indicated by decreased tumor burden in the treated mice compared to other groups revealing the efficiency of our rational.

3.2. Tumor growth biomarkers

In carcinogenesis, angiogenesis is essential for tumor progression and metastasis. Vascular endothelial growth factor (VEGF-1) is crucial in tumor angiogenesis, as it mediates the formation of the tumor blood vessels. Previous studies have reported the antiangiogenic effect of HK through down-regulation of the expression of VEGF-1 in cancer cells [52,76]. Herein, we have assessed the protein expression level of VEGF-1 in tumor tissue by ELISA (Fig. 5B). The group treated with HK-loaded LF-PNIPAM-co-AA(4:1) NGs P4 has showed reduction in VEGF-1 expression level by 2.89 and 3.28-folds in comparison with free HK-treated group and the positive control EAT-bearing mice, respectively.
Previous studies have proven that HK exerts its antitumor activity through inducing apoptosis via upregulating caspase-3 expression level [76,77]. Therefore, in our study, apoptosis was evaluated via estimating the caspase-3 protein expression level in tissue from EAT-bearing mice. Results showed that the treated groups displayed greater apoptotic activities with significantly elevated caspase-3 expression level compared to the positive control. The treated group with HK-loaded LF-PNIPAM-AA(4:1) NGs P4 demonstrated 1.97and 2.45-folds elevation in caspase-3 protein expression level in comparison with free HK-treated group and positive control, respectively (Fig. 5C). Furthermore, this result was confirmed via immunohistochemical analysis of tissue from EAT-bearing mice, which showed a significant (p < 0.05) elevation in number of caspase 3-positive immunostained cells in HK-treated (44.67 ± 2.6 %) and HK-loaded LF-PNIPAM-co-AA(4:1) NGs-treated group (92.33 ± 2.33 %) compared to positive control (11.33 ± 1.76 %) (Fig. 5D,E).

3.3. Histopathological and Ki67 immunohistochemical analysis of tumor tissue

Histopathological examination of solid mammary tumor excised from positive control, free HK, and HK-loaded LF-PNIPAM-co-AA(4:1) nanohydrogels-treated groups (P4) revealed circumscribed nodules of poorly differentiated viable and necrotic pleomorphic neoplastic cells (Fig. 6A). The viable neoplastic cells appeared with large hyperchromatic nucleus, prominent nucleolus, anisoneocleosis, and bipolar to multipolar mitotic division. In addition to apoptosis, HK has been reported to promote necrotic cell death in various types of cancer [78]. The semi-quantitative scoring of % necrosis in excised tumor tissue from positive control exhibited the lowest % necrosis (about 10 %) while free HK-treated group (about 25 %) (Fig. 6C). The highest % necrosis were found in tumor tissue from HK-loaded LF-PNIPAM-co-AA(4:1) nanohydrogels-treated groups (P4) (≥ 50 %). The proliferative activity was assessed via estimation of Ki-67 immunoexpression level in tissue from EAT-bearing mice (Fig. 6B). The results revealed that all HK-treated groups displayed lower proliferative activity in comparison with the positive control (74.33 ± 4.26 %). As a result of HK suppression of the expression level of VEGF-1 could enhance the inhibition of angiogenesis and proliferation. Tumor exercised from HK-loaded LF-PNIPAM-co-AA(4:1) nanohydrogels-treated group (P4) revealed significant decrease (p < 0.05) in Ki-67 expression level (21.33 ± 1.45 %) compared to free HK-treated group (61.67 ± 2.19 %) (Fig. 6D).

4. Conclusion

Herein, a dual stimuli responsive LF-PNIPAM-co-AA(4:1) copolymer encapsulating HK have been synthesized for breast cancer therapy. The prepared LF-PNIPAM-co-AA(4:1) NGs P4 displayed high % drug loading 18.65 wt.% with excellent physical stability. In vitro drug release of HK- loaded nanohydrogels exhibited significantly pH and temperature dependent behavior, a property which enhance the drug targeting delivery in tumor tissues thus minimizing the systemic side effects. In vitro cytotoxicity results demonstrated that IC50 of HK-loaded LF-PNIPAM-co- AA(4:1) NGs P4 was lower than free HK in both MCF-7 and A549 cancer cells. Moreover, the developed LF-PNIPAM-co-AA(4:1) NGs P3 have showed high cellular internalization by MCF-7 that increased with time, which could be correlated to the high binding capacity to (LFR1 and LFR2) receptors that overexpressed on cancer cell surface. In vivo, HK- loaded NGs exhibited reduction in tumor volume, which was accomplished by suppressing VEGF-1 and ki-67 expression levels that would inhibit the tumor proliferation. Moreover, HK-loaded NGs showed upregulation in the active caspse-3 expression level, therefore, inducing the apoptosis of tumor tissue in EAT-bearing mice. We can conclude that LF-targeted PNIPAM-co-AA(4:1) NGs P3 with both pH and temperature sensitivity presents a great potential nanocarrier for targeted cancer therapy.

5. Materials and methods

5.1. Materials

All the chemicals and instruments used were mentioned in the supplementary file.

5.2. Synthesis and physicochemical characterization

5.2.1. Preparation of PNIPAM-co-AA copolymer (P1, P2)

0.25 g (2.21 mmol) N-isopropylacrylamide, 0.050 g (0.137 mmol) sodium dodecyl sulfate (SDS), 0.010 g (0.045 mmol) N,N-methylene- bisacrylamide (BIS), and different amounts of acrylic acid were dissolved in double distilled water (15 mL) and stirred for 20 min under nitrogen at RT. Polymerization was initiated following addition of 0.020 g (0.061 mmol) potassium persulfate (KPS) and lasted for 4 h under nitrogen at 70 ± 2 ◦C. The copolymer was cooled down to room temperature and followed by extensive dialysis (Mwt cut off = 3.5 kDa) against double distilled water for 2 weeks. After dialysis, the copolymer solution was freeze dried to obtain instant water-soluble dry powder for further characterization [55].

5.2.2. Preparation of LF-PNIPAM-co-AA(4:1) nanohydrogels (P3)

PNIPAM-co-AA(4:1) copolymer (0.10 g) was dissolved in water (4.5 mL), then EDC.HCl (0.034 g, 0.18 mmol), K salt of oxyma (0.032 g, 0.18 mmol) and DMAP (0.022 g, 0.18 mmol) were added. The reaction mixture was preactivated for 10 min at RT. Lactoferrin (LF) (0.02 g, 2.5 × 10− 4 mmol) was dissolved in 5 mL water and added to the preactivated copolymer. The reaction mixture was stirred for 24 h at RT. After that the reaction mixture was dialyzed against deionized water for 48 h to get rid of byproducts. The resulting solution of LF-PNIPAM-co- AA NGs(4:1) (P3) was obtained as pale-yellow solution. The solution was lyophilized to obtain white powder.

5.2.3. Preparation of honokiol loaded LF-PNIPAM-AA(4:1) nanohydrogels (P4)

Honokiol (HK) was incorporated into the core of the LF-PNIPAM- AA(4:1) NGs by simple solvent evaporation method [79]. HK (24 mg) was dissolved in absolute ethanol (5 mL) and added drop by drop to the NGs solution of LF-PNIPAM-co-AA(4:1) (P3) (100 mg) in a rate 1 mL/min under low speed stirring and was left for 12 h until all the solvent was evaporated. The resultant HK loaded LF-PNIPAM-co-AA(4:1) NGs (P4) was purified by centrifugation at 3000 rpm for 10 min. Finally, the aqueous solution of HK/LF-PNIPAM-co-AA(4:1) NGs (P4) was lyophilized for further characterization.

5.2.4. Physiochemical characterization of PNIPAM-co-AA copolymers

The PNIPAM-co-AA (P1, P2) and LF-PNIPAM-co-AA(4:1) (P3) co-polymers were characterized using FT-IR and 1H-NMR spectroscopy to confirm the chemical structure changes that has been occurred. In addition, mass spectrometry was carried out on an AB/SCIEX 4800 Plus MALDI TOF/TOF (IET| International Equipment Trading Ltd., USA) to assess the change in molecular weights of the prepared copolymers. Furthermore, particle size, zeta potential, drug content, physical stability, morphological analysis, in vitro hemolysis and serum stability of the copolymers were performed as formerly illustrated and detailed in the Supplementary Material.

5.2.5. Lower critical solution temperature (LCST) analysis of synthesized nanohydrogels

The thermo-transition behavior of PNIPAM-co-AA(4:1) P2 and LF- PNIPAM-co-AA(4:1) P3 was evaluated by estimating the transmittance % of aqueous nanohydrogel solutions (5 mg/mL) with elevating temperature from 25 to 50 ◦C at 500 nm using an ultraviolet–visible spectrophotometer. The solution was incubated at each temperature in a waterbath at least 10 min to reach equilibrium temperature before data recording [64].

5.3. In vitro cytotoxicity uptake studies

The in vitro cell viability of free HK solution and honokiol loaded LF- PNIPAM-co-AA(4:1) NGs P4 against MCF-7 and A549 cells was estimated by MTT assay as formerly illustrated [41,80] and detailed in the Supplementary Material. The cellular uptake studies of RBITC-conjugated LF-PNIPAM-co-AA NGs into breast cancer cell line (MCF-7) was performed using confocal laser microscopy and flow cytometry (FACS Calibur, BD) as described in details in the Supplementary Material.

5.4. In vivo anti-tumor efficacy

The in vivo anti-tumor activity of HK-loaded LF-PNIPAM-co-AA(4:1) NGs was evaluated in comparison to free HK solution on female mice according to the standard protocol detailed in the Supplementary Material. Ehrlich ascites tumor (EAT) cells were utilized for tumor induction as illustrated in the Supplementary Material. Mice were divided into 4 groups (8 mice each) into negative control (healthy mice injected with saline), positive control (untreated EAT bearing mice), free HK-treated group, and HK-loaded LF-PNIPAM-co-AA(4:1) NGs-treated group. After tumor confirmation, the free drug or loaded LF-PNIPAM-co-AA(4:1) NGs were injected into the EAT bearing mice intravenously through the tail equivalent to 2.5 mg /kg HK daily injection for three weeks [81]. Mice were terminated via cervical dislocation after three weeks of treatment. The tumors were isolated and weighted then washed by cold saline and split into 2 parts. First part was stored at − 80 ◦C for further determination of tumor growth biomarkers. The second part was used for histopathological and immunohistochemical analysis via fixing in 10 % neutral buffered formalin followed by immersing in paraffin blocks. The experimental methods were detailed in the Supplementary Material.

5.5. Statistical analysis

Data analysis is detailed in the Supplementary Material.

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