3.1. Synthesis of PMT-ALG/LF-RST NHs F8
In this research, novel ALG/LF NHs were developed via chemical conjugation for the delivery of combined poorly soluble anticancer drugs, such as RST and HK, and the highly soluble cytotoxic drug PMT to breast cancer cells. ALG is a hydrophilic polysaccharide that exists in all types of brown algae. In addition to ALG biodegradability and biocompatibility, the rationale for choosing ALG as a nanoparticle shell is to exploit its stealth property to prevent reticuloendothelial recognition and subsequent removal, thus allowing passive accumulation of the nanoparticles in the tumor. Furthermore, lactoferrin (LF), a member of the Tf family, has established anticancer properties. Moreover, the rationale for choosing LF was to exploit its selective tumor-targeting action by binding to overexpressed multiple receptors in breast tumor cells. The conjugation of chemotherapeutic agents such as PMT and RST to ALG and LF, respectively, can enhance their efficacy and bioavailability and reduce side effects. Thus, RST conjugation to LF would improve its water solubility due to the hydrophilic nature of LF. Additionally, PMT conjugation to ALG would sustain its release in the systemic circulation, hence enabling its targeted delivery into tumor cells. In the current study, PMT-ALG/LF-RST NHs were developed through three steps. First, the LF-RST conjugate was synthesized via the formation of an amide bond via carbodiimide coupling. DIC/Oxyma was used to activate the carboxylic acid side chains of RST; thus, an intermediate Oxyma-activated ester molecule was formed and covalently coupled with the amine groups of LF [25-31]. The LF-RST conjugate showed a particle size of 179.0 nm with high RST loading (15.25 ± 0.56 wt.%) and ζ-potential of +14.7 mV. This is similar to a previous study reported by Abdelmoneem et al., which fabricated an LF-Celastrol (LF-CST) conjugate, where the hydrophilic property of LF was utilized to solubilize the hydrophobic drug.[24] Second, PMT-ALG conjugates were synthesized through ester bond formation by carbodiimide coupling. EDC.HCl/K. Oxyma was used to activate the carboxylic acid side chains of PMT to covalently couple with the hydroxyl groups of ALG. The resulting PMT-ALG conjugate showed a particle size of 267.9 nm with a high PMT loading (19.35± 0.64 wt.%) and ζ-potential of -47.1 mV [25-30]. Recently, polysaccharide drug conjugation was reported by Zhou et al., where dextran-RST was also prepared by a carbodiimide coupling reaction [32]. Third, PMT-ALG/LF-RST NHs were fabricated by coupling the PMT-ALG conjugate to the LF-RST conjugate through the formation of an amide bond by utilizing coupling reagents such as K. Oxyma and EDC. HCL, which enables the covalent coupling of free carboxylic acids of the PMT-ALG conjugate with the free amino groups of the LF-RST conjugate [25-30]. These newly synthesized nanohybrids can self-assemble into spherical nanohybrids consisting of an LF-RST conjugate inner core as a reservoir for hydrophobic drugs and a PMT-ALG conjugate as a hydrophilic shell, where conjugation of RST to the LF polymer could also increase the hydrophobicity of LF [33] [34]. In our preliminary study, two conjugation ratios between ALG and LF were investigated for the preparation of ALG/LF NHs (Table 1). The ALG:LF (1:2) ratio was finally selected based on zeta potential and particle size characterization. The resultant PMT-ALG/LF-RST NHs exhibited a particle size of 304.9 nm and a greatly negative ζ-potential of -43.8 mV, which might correspond to the free COOH groups of ALG on the surface of the copolymer (Table 1). The preparation steps of crosslinked HK-loaded PMT-ALG/LF-RST NHs are illustrated in the schematic diagram shown in Fig. 1.
Table 1. Physicochemical Characteristics and Composition of Crosslinked HK/PMT-ALG-LF-RST NHs. Zeta potential, particle size, entrapment efficiency (EE), drug loading (DL), and conjugation efficiency (CE %) of NPs (n=3):
|
Formula
|
Particle size (nm)
|
PDI
|
ζ-potential (mV
|
RST
|
PMT
|
HK
|
DL
(mg/wt.%)
|
%CE
|
DL
(mg/wt.%)
|
%CE
|
DL
(mg/wt.%)
|
%EE
|
F1
|
ALG/LF (1:1)
|
220.6±1.8
|
0.383
|
-47.1±0.41
|
|
|
-
|
-
|
-
|
-
|
F1
|
ALG/LF (1:2)
|
163.9±2.3
|
0.342
|
-41.1±0.53
|
-
|
-
|
-
|
-
|
-
|
-
|
F2
|
LF-RST
|
179.0±1.3
|
0.371
|
+14.7±0.91
|
16/13.79
|
64.0
|
-
|
-
|
-
|
-
|
F3
|
ALG/LF-RST
|
239.0±1.8
|
0.348
|
-43.8±0.27
|
16/9.63
|
64.0
|
-
|
-
|
-
|
-
|
F4
|
HK-loaded ALG/LF RST
|
365.2±2.1
|
0.347
|
-46.9±0.65
|
16/8.89
|
64.0
|
-
|
-
|
14/7.69
|
93.3
|
F5
|
ALG-PMT
|
267.9±1.2
|
0.458
|
-47.1±0.72
|
-
|
-
|
12/19.35
|
80.0
|
-
|
-
|
F6
|
PMT-ALG/LF
|
224.8±1.6
|
0.367
|
-39.7±0.39
|
-
|
-
|
12/7.40
|
80.0
|
-
|
-
|
F7
|
HK-loaded PMT-ALG/LF
|
333.0±1.9
|
0.410
|
-41.2±0.83
|
-
|
-
|
12/6.81
|
80.0
|
14/7.95
|
93.3
|
F8
|
PMT-ALG/LF-RST
|
304.9±2.7
|
0.464
|
-43.8±0.56
|
16/8.98
|
64.0
|
12/6.67
|
80.0
|
-
|
-
|
F9
|
Uncrosslinked HK loaded PMT-ALG/LF-RST
|
389.7±1.5
|
0.423
|
-44.7±0.32
|
16/8.33
|
64.0
|
12/6.18
|
80.0
|
14/7.21
|
93.3
|
F10
|
Crosslinked HK-loaded PMT-ALG/LF-RST
|
258.7±0.95
|
0.342
|
-45.3±0.47
|
16/7.05
|
64.0
|
12/5.24
|
80.0
|
14/6.11
|
93.3
|
The conjugation reactions were confirmed by 1H-NMR analysis (Fig. 2). The 1H-NMR spectrum of LF in DMSO-d6 (Fig. 2B) shows multiplet peaks at the variation between 0.50 and 2.40 ppm attributed to the peptide chain aliphatic protons of LF. Moreover, two broad multiplet peaks were observed at 6.60-6.70 and 7.10-7.30 ppm, attributed to the peptide chain aromatic protons and NH protons. The 1H-NMR spectrum of the LF-RST conjugate (Fig. 2A) reveals a broad singlet peak at 1.25 ppm attributed to the methyl groups of RST. In addition, 2 singlet peaks at 2.74 and 2.91 ppm corresponding to the S-CH3 and N-CH3 groups of RST, respectively, were observed. Additionally, multiplet peaks attributed to the aromatic protons of RST are observed in the range of 6.90-8.00 ppm. On the other hand, the 1H-NMR spectrum of ALG in D2O (Fig. 2E) shows multiplet peaks in the range 3.64-4.16 ppm. The 1H-NMR spectrum of the ALG-PMT conjugate in D2O (Fig. 2D) shows peaks in the range of 1.70-3.10 ppm, characteristic of the methylene protons of PMT. Moreover, multiplet peaks were observed in the range of 7.81-7.82 ppm related to the aromatic protons of PMT. The 1H-NMR spectrum of PMT-ALG-LF-RST NHs (Fig. 2C) shows 3 multiplet peaks in the range of 0.80-1.23 ppm attributed to the aliphatic protons of the LF-RST conjugate. Furthermore, peaks in the range of 1.70-3.10 ppm were observed to be characteristic of the methylene protons of PMT. Moreover, multiplet peaks attributed to the alginate aliphatic C-H protons were observed in the range of 3.64-4.00 ppm.
3.2. Development of crosslinked HK-loaded PMT-ALG/LF-RST NHs
In contrast to PMT and RST, which were covalently coupled to the ALG-LF backbone, HK was physically loaded inside the hydrophobic core of PMT-ALG/LF-RST NHs via a simple solvent evaporation method. There may be an abundance of co-acting intermolecular interactions between the carrier material and the drug in the loading process, such as van der Waal forces, hydrophobic interactions and hydrogen bonding. All of these forces can play a role in effective HK loading and nanohybrid stabilization [35, 36]. Finally, the crosslinking of polymeric nanohybrids with genipin seems to be an excellent strategy to enhance their structural stability and prevent rapid drug release and premature disintegration. This study revealed that genipin successfully crosslinked the amine groups of LF with a significant reduction in the nanohybrid size and drug release profile. The crosslinking reaction by genipin led to the appearance of an intense blue color. Upon crosslinking of nanohybrids, the particle size markedly decreased from 389 nm to 258.7 nm by virtue of forming more compact and denser nanohybrids (Table 1 and Fig. 3A, B) [37]. During our preliminary investigations, different amounts of genipin were used for the crosslinking of ALG-LF nanohybrids (Table 2). Approximately 35 mg (1:5.54 wt. ratio) of genipin was finally selected based on PS and PDI characterization.
Table 2. Effect of genipin amounts on zeta potential, PDI and particle size of nanohybrids F10 (n=3):
Amount of genipin (mg)
|
F9: genipin
wt. ratio
|
particle size (nm)
|
PDI
|
10
|
1:19.4
|
389.0±0.30
|
0.450
|
20
|
1:9.7
|
320.0±0.60
|
0.410
|
30
|
1:6.46
|
290.0±0.87
|
0.360
|
35
|
1:5.54
|
258.7±0.95
|
0.342
|
50
|
1:3.88
|
400.0±1.20
|
0.420
|
* The amount of HK-loaded PMT-ALG/LF-RST NHs F9 used is 194 mg.
3.3. Solid-State Characterization.
The FT-IR spectra of our prepared formulations were used to study the chemical modification (Fig. 3C). The FT-IR spectrum of the LF-RST conjugate shows two bands at 1317 and 1151 cm−1 attributed to the SO2 group of RST. Additionally, a band at 1540 cm-1 specific to the C=N group of RST was observed. Furthermore, LF characteristic absorption bands ranging between 3600-2600 cm-1 are assigned to N-H and hydroxyl groups, where bands at 2961 cm-1 and 2865 cm-1 assigned to the sp3 C-H stretching vibration were observed. Additionally, the most distinctive bands of LF at 1651 (amide I) and 1448 (amide II) cm−1 were observed. The new amidic carbonyl group in the LF-RST conjugate overlapped with that of LF at 1651 cm−1, which confirms amide bond formation between LF and RST. On the other hand, the FT-IR spectrum of the ALG-PMT conjugate (Fig. 3C) showed a broad stretching band in the range 3300 to 3000 cm−1 attributed to the hydroxyl group of ALG. Furthermore, the disappearance of the strong band of PMT at 1690 cm-1 and the broad band ranging between 3600-2500 cm-1 attributed to carboxylic acid confirms PMT conjugation. Moreover, a stretching band at 1701 cm-1 related to the new ester carbonyl group in the PMT-ALG conjugate was observed.[33] In addition, the FT-IR spectrum of PMT-ALG-LF-RST NHs (Fig. 3C) shows an absorption band at 1652 cm-1, corresponding to the new amide carbonyl group of the nanohybrids overlapped with that of the LF and LF-RST conjugates at 1651 cm−1. Moreover, an absorption band at 1302 cm−1 attributed to the SO2 group of RST was observed. The band at 1542 cm-1 related to the C=N group of RST was also noticed. In addition, the FT-IR spectrum of HK-loaded PMT-ALG/LF-RST NHs reveals a broad absorption band at 3292 cm−1, which is related to the OH group of HK. This band overlapped with the broad band between 3600-2500 cm-1 corresponding to N-H and hydroxyl groups characteristic of LF and the hydroxyl group corresponding to ALG. Moreover, two absorption bands at 1639 cm−1 and 1426 cm−1 assigned to the phenyl ring of HK were observed. This absorption band (1639 cm−1) is overlapped by the amidic carbonyl group of the copolymer at 1652 cm-1. Additionally, an absorption band at 3084 cm-1 attributed to the sp2 C-H stretching band of HK was observed. The absorption band at 1217 cm-1 related to the C-O bond of HK was also observed. The FT-IR spectrum of crosslinked HK-loaded PMT-ALG/LF-RST NHs (Fig. 3C) shows the characteristic absorption bands of genipin, which appear in the fingerprint region of the spectrum. Moreover, a very broad absorption band between 3600-2600 cm-1 was attributed to N-H and hydroxyl groups characteristic of LF, and the hydroxyl groups of ALG were observed.
The DSC thermograms of RST revealed endothermic peaks at approximately 80°C and 164°C, which were assigned to the drug melting temperature (Fig 4A) [38]. The thermogram of PMT showed three distinctive endothermic peaks at 91.78, 153.82 and 243.80°C [39]. The characteristic peaks of RST and PMT are not observed in the PMT-ALG/LF-RST NHs F8 thermogram, which emphasizes the amorphous nature of these NHs[24]. Additionally, the natural state of HK exists in a crystalline form and reveals its melting peak at approximately 72.43°C. The DSC thermogram of HK-loaded PMT-ALG/LF-RST NHs F9 showed only an endothermic peak at 341.29°C, confirming the loading of HK within NHs in an amorphous form [40].
3.4. Morphological analysis, physical stability and redispersibility
The TEM micrograph of crosslinked HK-loaded PMT-ALG/LF-RST NHs F10 showed a spherical shape with a diameter range of 141-233 nm with no agglomerated particles, confirming their elevated colloidal stabilization (Fig. 4B). The TEM images also exhibited the formation of a distinctive core-shell structure composed of the hydrophilic corona of PMT-ALG surrounding the hydrophobic core of LF-RST. It was also noticed that after storage for 3 months at 4°C, both HK-loaded PMT-ALG/LF-RST NHs F9 and crosslinked HK-loaded PMT-ALG/LF-RST NHs F10 maintained their PSs of 410±1.9 and 268 ±0.3 nm, respectively, without a significant difference from the NHs that were initially stored, indicating their fair stability (Fig. 4C). The high zeta potential (-45.3 and -44.7 mV) of both F9 and F10 NHs may explain their great stability, as ALG negatively charged side chains induce strong repulsive forces between NHs. In addition to the repulsion mechanism, stabilization of the NHs may be enhanced by the glycan chain of LF by improving the interdomain interactions of LF and protecting against protein degradation [41, 42]. The physical stability of the NHs can be further improved by lyophilization of the prepared NHs into a dry powder [43]. In our research, no cryoprotectant was needed, and a fluffy powder was obtained that could be redispersed in H2O, forming a colloidal solution with no aggregation. The reconstituted lyophilized F9 and F10 NHs demonstrated PS of 380±0.8 and 250±0.8 nm with redispersibility index values of 0.966 and 0.975, respectively, where values less than 1.0 are considered efficient [44, 45]. Furthermore, the zeta potential of the NHs after lyophilization did not markedly change (Table 3).
Table 3. Freeze-drying effect on the PS, zeta potential and yield of F9 and F10 NHs (n=3):
Formula
|
Yield (% w/w)
|
PS (nm)
|
RI* (Sf/Si)
|
ζ-potential (mV)
|
Before
|
After
|
Before
|
After
|
HK-loaded PMT-ALG/LF-RST NHs F9
|
92.3%
|
389.7±0.5
|
380.0±1.2
|
0.975
|
-44.7±1.3
|
-45.0±0.6
|
Crosslinked HK-loaded PMT-ALG/LF-RST NHs F10
|
94.4%
|
258.7±0.9
|
250.0±0.8
|
0.966
|
-45.3±0.5
|
-46.1±0.7
|
* RI: Redispersibility index (Final particle size / Initial particle size)
3.5. In vitro drug release:
The in vitro release of PMT, RST and HK from uncrosslinked HK-loaded PMT-ALG/LF-RST NHs F9 and crosslinked HK-loaded PMT-ALG/LF-RST NHs F10 was evaluated at pH 4, 5.5 and 7.4 using the dialysis method in PBS (Fig. 5 B). A, B). The results revealed that HK release from NHs at pH 4, 5.5 and 7.4 did not differ significantly. The release profile of HK loaded in NHs was biphasic with a fast release during the first 8 h (approximately 30% and 18.5% from F9 and F10 NHs, respectively) followed by slow release (with approximately 55.5% and 34% from F9 and F10 NHs, respectively) for the remaining 120 h. Early rapid release can be ascribed to the fact that part of the drug is localized at the shell or the core-shell interface, but the slow-release phase of the drug can be due to that part of the drug entrapped physically in the hydrophobic core of the nanohybrids [46]. Typically, the release rate of HK from crosslinked NHs F10 is slower than the rate from uncrosslinked NHs F9, as the degree of nanostructural tortuosity was enhanced and the space between polymer chains was reduced by crosslinking [47]. Unlike HK, the results showed that crosslinking had no effect on PMT release. PMT showed a sustained release from the NHs at pH 4, reaching 40% over 5 days as the ester bond can be hydrolyzed in acidic medium, while the release decreased at pH 5.5 (approximately 5%), and no release was detected at pH 7.4 over the entire period of the experiment. The PMT release was very low due to the need for ester bond cleavage for enzymatic or chemical degradation. Our results are consistent with the previously mentioned in vitro drug release investigation, which was performed on DTX-polymer conjugate, where the conjugate released approximately 15% over 20 days of DTX under physiological circumstances (pH 7.4) and slightly higher in acidic environments [48]. On the other hand, even after long-term incubation at acidic pH, RST release from F9 and F10 NHs could not be detected. This is expected for RST, which was coupled by a highly stable amide bond that would only be cleavable inside the cancer cells under the effect of the endosomal enzymes. Markovsky et al. reported similar results after extended incubation at acidic pH, where no in vitro release of doxorubicin from PGA–paclitaxel–doxorubicin conjugate was observed [49]. The slow release of the drug from the developed NHs would enable them for parenteral administration due to their stability at physiological pH, allowing improved drug accumulation and localized drug release at the site of the tumors [50].
3.6. Hemocompatibility and serum stability
The stabilization of intravenous nanoformulations in serum is important in their drug delivery application. Nanohybrids F9 and F10 showed no significant change in their particle size (from 389.7 ±0.5 to 396 ±1.2 nm and from 258.7±0.95 to 260 ±1.7 nm, respectively) when mixed with fetal bovine serum (FBS) (Fig. 5C). This could be attributed to the hydrophilic brush-like structure of the ALG shell of NHs that leads to minimal protein adsorption on NHs, in addition to the hydrophobic core protection from biological invasion [51] and surface passivation of nanohybrids. Elevated stability of NHs in serum might be due to repulsion force between the serum proteins having negative charges and the prepared nanohybrids. After incubation for 4 h with FBS, the particle sizes of F9 and F10 NHs reached 407 ±0.2 nm and 275 ±1.2 nm, respectively, which decreased to 396 ±1.2 nm and 260 ±1.7 nm after 6 h. This action might be attributed to protein molecule association and dissociation on the NH surface through incubation [52].
On another avenue, the hemolytic activity of F9 and F10 NHs was approximately 3.7% and 3.3%, respectively, up to a 1 mg/mL concentration (Fig. 5D, E). In general, the nontoxic and safe percentage of hemolytic activity is less than 5% [53]. Our prepared NHs exhibited acceptable hemolytic activity by virtue of surface passivation of NHs by incorporation of ALG polymer to suppress protein and cell attachment to the surface of NHs [54]. These results indicated that F9 and F10 NHs have good hemocompatibility and are suitable for parenteral administration.
3.7. In vitro cytotoxicity
The efficiency of the free PMT, free RST, free HK, free dual combinations (RST/HK, PMT/RST, PMT/HK) and free triple combinations (PMT/RST/HK) against cancer was studied on MCF-7 breast cancer cells compared to the developed nanohybrids after 24 h of exposure using the MTT assay (Fig. 6A, B). First, the noncytotoxicity of blank ALG/LF NHs against MCF-7 cells after 24 h was confirmed, indicating their safety and biocompatibility (IC50=3095.443). Compared to free single and free dual drugs, free combination therapy (PMT/RST/HK) displayed higher cytotoxicity, revealing the synergistic effect between the three drugs. Regarding the nanohybrids, it seemed that dual drug-loaded nanohybrids (HK loaded-ALG/LF-RST NHs F4, PMT-ALG/LF-RST NHs F8 and HK loaded-PMT-ALG/LF NHs F7) improved the potency of the combination, showing IC50 values with 0.61-, 0.46-, and 0.44-fold reductions compared to free combination therapy (PMT/RST/HK), respectively. On the other hand, the crosslinked HK/PMT-ALG-LF-RST NHs F10 revealed the minimum IC50 value in comparison to the other prepared NHs.
CompuSyn software mentioned by Chou and Talalay was utilized to perform more extensive statistical analysis [55-57]. The dose reduction index (DRI) and combination index (CI) were estimated to assess the antitumor efficiency of the prepared NHs relative to the free combination therapy (Table 4). The outputs showed that, compared to the free drug combination, all prepared NHs had higher anticancer activity, especially uncrosslinked and crosslinked HK-loaded PMT-ALG/LF-RST NHs F9 and F10, where their CIs were 0.0556 and 0.0336, respectively, supporting the synergism accomplished by triple loading of PMT/RST/HK in NHs. Moreover, the dose reduction index (DRIs) of PMT were 51.22 and 84.84 in F9 and F10 NHs, respectively. The RST DRIs were 36.85 and 61.04 in the F9 and F10 NHs, respectively. The DRIs of HK were 111.3 and 184.34 in the F9 and F10 NHs, respectively.
Table 4. CI, IC50, and DRI values of free drugs in comparison to the synthesized NHs against MCF-7 breast cancer cells after 24 h at concentrations of 0-100 μM
Compound
|
CI value
|
Total IC50 of Combination
|
Dose PMT
|
Dose RST
|
Dose HK
|
DRI of PMT
|
DRI of RST
|
DRI of HK
|
HK
|
-
|
54.95
|
-
|
-
|
-
|
-
|
-
|
-
|
RST
|
-
|
27.93
|
-
|
-
|
-
|
-
|
-
|
-
|
PMT
|
-
|
19.22
|
-
|
-
|
-
|
-
|
-
|
-
|
RST/HK
|
0.691
|
23.31
|
-
|
14.52
|
9.08
|
-
|
1.93
|
5.81
|
PMT/RST
|
0.672
|
16.31
|
5.46
|
10.93
|
-
|
3.55
|
2.56
|
-
|
PMT/HK
|
0.481
|
14.18
|
6.40
|
|
8.00
|
3.03
|
|
6.58
|
PMT/RST/HK
|
0.286
|
7.97
|
1.96
|
3.91
|
2.44
|
9.94
|
7.15
|
21.59
|
HK-loaded ALG/LF-RST NHs F4
|
0.142
|
4.90
|
-
|
2.99
|
1.86
|
-
|
9.36
|
28.26
|
HK-loaded PMT-ALG/LF NHs F7
|
0.114
|
3.50
|
1.52
|
-
|
1.91
|
12.73
|
-
|
27.66
|
PMT-ALG/LF-RST NHs F8
|
0.149
|
3.65
|
1.21
|
2.43
|
-
|
16.00
|
11.51
|
-
|
HK-loaded PMT-ALG/LF-RST NHs F9
|
0.055
|
1.63
|
0.38
|
0.76
|
0.47
|
51.22
|
36.86
|
111.30
|
Crosslinked HK-loaded PMT-ALG/LF-RST NHs F10
|
0.033
|
0.94
|
0.23
|
0.46
|
0.29
|
84.84
|
61.04
|
184.30
|
3.8. In vitro cellular uptake of nanohybrids:
For nanohybrid fluorescent labeling, the LF core of the nanohybrids was conjugated to the thiocyanate group of RBITC dye via its free amino groups. Confocal microscopy was utilized to evaluate the uptake of RBITC-labeled uncrosslinked PMT-ALG/LF-RST F8 and crosslinked PMT-ALG/LF-RST NHs after incubation with MCF-7 cells at 37°C for 4 h and 24 h (Fig. 7A). Our results revealed that crosslinked F8 NHs exhibited greater cellular uptake efficacy in comparison to uncrosslinked F8 NHs, as suggested by the powerful intensity of red fluorescence noticed in cells treated with the former. This could be ascribed to the lower particle size of crosslinked F8 facilitating its cellular internalization [58]. The intensity of fluorescence for both NHs increased after 24 h of incubation, suggesting that the process of cellular internalization of the prepared nanohybrids is time-dependent. LF actively targets cancer cells through its interaction with LF receptors that are overexpressed on the cancer cell surface and improve the cellular uptake of nanohybrids [59]. The proton sponge effect and swelling properties of ALG have been revealed to mediate cellular uptake [58]. Flow cytometry analysis confirmed the reliability and accuracy of the results, where comparing the fluorescent intensity of cells treated with uncrosslinked F8 NHs to those treated with crosslinked F8 NHs indicated much greater cellular uptake of the crosslinked F8 after 4 h of incubation with MCF-7 cells, as revealed in Fig 7B, C.
3.9. In vivo antitumor efficacy
3.9.1. Tumor growth
The in vivo antitumor effect for crosslinked HK-loaded PMT-ALG/LF-RST NHs F10 compared with free HK, free RST, free PMT and free (HK/RST/PMT) combination treatment was investigated using mice bearing Ehrlich ascites tumors (EAT). The treatment of mouse groups bearing EAT was conducted for three consecutive weeks while monitoring the tumor size during this period. Following treatment, the highest elevation in the tumor size percentage was in the positive control group, which reached 587%. This was higher than those detected in the free HK (205%), free RST (183%), free PMT (177%), free (HK/RST/PMT) combination therapy (125%) and crosslinked HK-loaded PMT-ALG/LF-RST-treated groups (103%) (Fig. 8A). Obviously, the greatest anticancer activity was exhibited by crosslinked HK-loaded PMT-ALG/LF-RST NHs F10, as the tumor burden was reduced in the treated mice in comparison to other groups, showing the efficacy of our rationale.
3.9.2. Biomarkers of Tumor Growth
Angiogenesis plays a pivotal role in tumor metastasis and progression. Vascular endothelial growth factor (VEGF-1) is a critical factor in tumor angiogenesis. Recently, some investigations have mentioned the antiangiogenic influence of PMT, RST and HK by downregulation of VEGF-1 expression in tumor cells [60-63]. Herein, ELISA was used to evaluate the degree of VEGF-1 protein expression in tumor tissue (Fig. 8B). Using our prepared crosslinked HK loaded-PMT-ALG/LF-RST NHs F10, VEGF levels were reduced successfully by 2.596-fold, while the free HK/PMT/RST combination reduced VEGF levels only by 1.744-fold compared to the positive control.
Recent investigations have reported apoptotic effects induced by HK and PMT through the upregulation of caspase-3 expression [62, 64, 65]. In the current investigation, the caspase 3 expression level was estimated in tissue from EAT-bearing mice to evaluate the apoptotic effect. The results revealed that the apoptotic activity in the treated groups was greater than that in the positive control with a considerably elevated caspase-3 expression level. Our prepared crosslinked HK/PMT-ALG-LF-RST NHs F10 succeeded in elevating caspase-3 protein expression levels by 2.769-fold versus only a 1.659-fold increase for free HK/PMT/RST combination therapy in comparison to the positive control (Fig. 8C). Moreover, immunohistochemical investigation of mice bearing Ehrlich ascites tumor (EAT) confirmed our result, which revealed a marked (p < 0.05) increase in the count of caspase 3-positive immune stained cells in HK-treated (37.67±2.33), RST-treated (38.67±1.45), PMT-treated (45.00±4.04), free HK/PMT/RST combination (74.00±3.21), and crosslinked HK loaded PMT-ALG/LF-RST NHs F10 (92.00±1.73) mice compared with untreated positive control (9.33±0.88) mice (Fig. 8D, E).
3.9.3. Histopathological investigation and Ki67 detection in cancerous tissue
In untreated positive control mice, the solid mammary tumor showed circumscribed nodules of necrotic pleomorphic neoplastic and poorly differentiated viable cells. The viable neoplastic cells were characterized by prominent, large hyperchromatic nuclei, anisonucleosis, and bipolar to multipolar mitotic division. However, mice treated with free HK, free RST, free PMT, free PMT/HK/RST combination and crosslinked HK loaded-PMT-ALG/LF-RST NHs F10 revealed similar histologic characterization of neoplastic cells with different degrees of necrosis (Fig. 9A). Moreover, HK and PMT have been reported to enhance the death of necrotic cells in different kinds of cancer [66, 67]. Necrosis scored semiquantitatively in each excised tumor exhibited a significant elevation in the expression percentage in free HK-treated (approximately 25%), free RST-treated (approximately 25%), free PMT-treated (approximately 25%), free PMT/HK/RST combination (approximately 35%), and crosslinked HK-loaded PMT-ALG/LF-RST NHs F10 (≥ 50%) mice compared with untreated control positive mice (approximately 10%) (Fig. 9B). The degree of Ki-67 immunoexpression in EAT mice was evaluated to assess proliferative activity (Fig. 9C). The proliferation rate was represented by the significant (p < 0.05) decrease in the count of Ki67-immunoreactive cells in HK-treated (46.00±2.65), RST-treated (48.33±2.33), PMT-treated (40.33±2.03), free (PMT/HK/RST) combination (26.67±5.04), and crosslinked HK loaded-PMT-ALG/LF-RST NHs F10 (17.67±2.33) mice compared with untreated positive control (83.67±3.38) rats (Fig. 9D). PMT and RST have been reported to lower the density of the tumor cell proliferation protein Ki-67 [68, 69].