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. 2023 Feb 2;8(6):5655–5671. doi: 10.1021/acsomega.2c07128

Engineered Sericin-Tagged Layered Double Hydroxides for Combined Delivery of Pemetrexed and ZnO Quantum Dots as Biocompatible Cancer Nanotheranostics

Riham M Abdelgalil †,‡, Sherine N Khattab §,*, Shaker Ebrahim ∥, Kadria A Elkhodairy †,‡, Mohamed Teleb ‡,⊥, Adnan A Bekhit ‡,⊥,#, Marwa A Sallam †, Ahmed O Elzoghby †,‡,∇,*
PMCID: PMC9933221  PMID: 36816638

Abstract

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Aim: Despite extensive progress in the field of cancer nanotheranostics, clinical development of biocompatible theranostic nanomedicine remains a formidable challenge. Herein, we engineered biocompatible silk-sericin-tagged inorganic nanohybrids for efficient treatment and imaging of cancer cells. The developed nanocarriers are anticipated to overcome the premature release of the chemotherapeutic drug pemetrexed (PMX), enhance the colloidal stability of layered double hydroxides (LDHs), and maintain the luminescence properties of ZnO quantum dots (QDs). Materials and Methods: PMX-intercalated LDHs were modified with sericin and coupled to ZnO QDs for therapy and imaging of breast cancer cells. Results: The optimized nanomedicine demonstrated a sustained release profile of PMX, and high cytotoxicity against MDA-MB-231 cells compared to free PMX. In addition, high cellular uptake of the engineered nanocarriers into MDA-MB-231 breast cancer cells was accomplished. Conclusions: Conclusively, the LDH-sericin nanohybrids loaded with PMX and conjugated to ZnO QDs offered a promising cancer theranostic nanomedicine.

1. Introduction

According to GLOBOCAN 2020, with an anticipated 2.3 million laterally cases every year, the rate incidence of the female breast cancer (11.7%) recently exceeded the rate of lung cancer (11.4%).1 Limitations of existing cancer imaging modalities urged the fabrication of novel imaging probes with better sensitivity and specific profile.2 Quantum dots (QDs) are semiconductor crystals of nanometer size with tunable sizes that range from 2 to 10 nm.3 Their unique optical attributes, including those of high quantum yield, size-tunable light emission, good chemical, and photostability, help to alleviate the limitations concerned with organic dyes or other imaging modalities.3 They are also able to emit in both near-infrared (NIR) and visible regions of light that are favorable for various biological applications.3 Unfortunately, the toxicity related to QDs, especially Cd-based QDs, is a major concern in biological applications.3 Hence, it was necessary to develop Cd-free QDs such as ZnO QDs to be nontoxic and biodegradable, in addition to their magnificent photoluminescence (PL) properties that suit biological imaging.4 Nanotheranostics is indeed a term pointing to the use of nanotechnology for therapy (drugs, or photothermal therapy), and diagnosis.5 Organic nanocarriers fabricated from natural polymers including proteins and polysaccharides can be implemented as ideal drug carriers for various therapeutic and diagnostic agents.6−10 Sericin is a natural glue-like protein recovered from cocoons of silkworms that entraps a protein core, silk fibroin, and contains 18 amino acids.11 Sericin, derived from Bombyx mori silkworm cocoons, has around 200 kDa molecular weight. Aspartic acid (16%), serine (37%), glycine, glutamic acid, threonine, and tyrosine are the predominant essential amino acids. Actually, the existence of different chemical functional groups like hydroxyl, carboxyl, and amino groups enables the reaction with various drugs and polymers to cope with various medical applications.11 Silk sericin possesses different biological activities that are useful in different biomedical aspects like anti-elastase, anti-tyrosinase, and moisturizing effects.12 Sericin can also operate as a pro-oxidant since it contains polyhydroxy amino acids (like serine) along with polyphenols and flavonoids as secondary metabolites. By eliciting a redox imbalance, A431, SAS, and MCF-7 cancer cells were found to be suppressed by sericin recovered from the cocoons of Antheraea assamensis, B. mori, and Philosamia ricini.13 The highly hydrophilic nature of sericin greatly encourages the fabrication of sericin nanoparticles (NPs) using the well-defined desolvation technique with subsequent crosslinking to entrap various drugs or other NPs for different drug delivery applications, providing a sustained release profile of the entrapped drugs besides the high entrapment efficiency.14,15 Especially in cancer therapy, efficient cellular uptake can be achieved through adjustable morphology and nanosize via passive targeting and enhanced permeation and retention (EPR) effect.16

Layered double hydroxides (LDHs), a well-known lamellar inorganic drug delivery system, have a two-dimensional (2D) structure and belong to the class of hydrotalcite or anionic clays. The believed high biocompatibility, loading capacity, and anionic exchange properties, besides the ease of synthesis, allow them to have enormous potential as drug delivery agents. Also, the controlled and pH-dependent release profile of LDH is advantageous in cancer drug delivery.17 Moreover, the general structure of LDH is [M1–x2+Mx3+(OH)2]x+(A–m)x/m·nH2O, where M2+ is a divalent cation, M3+ is a trivalent cation, A is a counter anion with a negative charge (m), and x is the mole fraction of M3+, which equals to [M3+/(M2+ + M3+)].17 The cytotoxicity of LDH NPs is reported to be lower than that of other inorganic NPs. Moreover, LDH are reported to exhibit significantly higher cytotoxicity against cancer cells compared to normal cells. In addition, LDH appears to have no acute toxicity in vivo when administered at a practical dosage for biomedical purposes. LDH NPs dissolve within 24 h and do not concentrate in any one organ. This evidence reveals that LDH is a suitable nanocarrier with high biocompatibility and biosafety.18

Alimta (pemetrexed, PMX, Eli Lilly and Company, Indianapolis, Indiana) is an antimetabolite that acts by repressing three folate-dependent enzymes known as thymidylate synthase (TS), dihydrofolate reductase (DHFR), and glycinamide ribonucleotide formyltransferase (GARFT) that eventually interfere with RNA and DNA biosynthesis.19 PMX is believed to be the primary antifolate drug endorsed for malignant pleural mesothelioma (MPM) treatment. Furthermore, PMX has been contemplated as a solitary anticancer agent and in combined regimens for patients suffering from neck malignancies, genitourinary, colorectal, gastric, pancreatic, and breast cancer.20 Overcoming formulation constraints, minimizing side effects, and improving therapeutic efficacy can all be achieved through encapsulating PMX into an adequate drug delivery system, so it appears to be a potential technique for this drug.

In this study, we propose the employment of modified inorganic nanocarriers LDHs with sericin protein by an established desolvation technique for the first time up to our knowledge, thus combining the advantages of both protein and inorganic nanocarriers, followed by their chemical conjugation to ZnO capped by 3-triethoxysilylpropan-1-amine (APTES-ZnO) QDs as a nanodelivery system for the hydrophilic PMX drug. LDHs are modified with sericin to add additive sustained release profile of PMX. In addition, its biocompatibility and biosafety in terms of immunogenicity and inflammation encourage its use in different biomedical aspects, especially nanocarrier fabrication. In addition to its modifiable structure and excellent biological characteristics such as amphiphilicity, pH responsiveness, and biodegradability, sericin contains abundant polar amino acids, which can interact with water molecules to form a hydration layer. Thus, sericin is possible to be used as a dispersion stabilizer to enhance the stability and hemocompatibility of NPs after their coating.21 This work is based on the optimization of each step in the formulation to obtain the most appropriate drug delivery system for the proposed application. It comprehended the effect of guest anion concentration on its behavior, whether it is adsorbed on the surfaces of LDH or intercalated within the brucite layers. Besides, the crosslinker effect on the luminescence properties of the employed ZnO QDs is studied. Moreover, this formulation is intended to be employed as a theranostic drug delivery system to diagnose and treat breast cancer. It enables monitoring the diseased tissues, anticancer efficacy, and subsequently provides the chance to tune the therapeutic strategy in a controlled and rational manner. Distinctly, this formulation can aid in the tailoring of personalized medicine.

2. Results and Discussion

2.1. Synthesis and Characterization of APTES-ZnO QDs

As illustrated in Figure 1a, ZnO QDs were synthesized utilizing the sol–gel process in this study. Such a technique has various merits including simplicity, low cost, high yield of relatively homogeneous small-sized QDs, and low-temperature synthesis.22 Surface salinization is a promising strategy to stabilize ZnO nanocrystals and prevent their decomposition in aqueous media. Therefore, trialkoxysilanes can form strong covalent bonds with ZnO hydroxyl groups, producing a crosslinked polysiloxane shielding barrier. 3-Triethoxysilylpropan-1-amine (APTES) was used as a capping agent, which provided amino functional groups to be further conjugated to biomolecules.22

Figure 1.

Figure 1

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(a) Synthesis of APTES-ZnO QDs, (b) UV–visible absorption spectrum of APTES-ZnO QDs, (c) photoluminescence (PL) emission spectrum of APTES-ZnO QDs upon excitation at λ = 330 nm, (d) ζ-potential of APTES-ZnO QDs, and (e) high-resolution transmission electron microscopy (HRTEM) image of APTES-ZnO QDs.

The synthesized QDs showed a broad absorption peak at 315 nm (Figure 1b). Moreover, a broad emission peak was detected at ∼533 nm in the visible region upon excitation at 330 nm. In addition, a narrow emission peak appeared at 395 nm in the UV region (Figure 1c), as previously reported.23,24 The broad bandwidth of ZnO QDs implied that the PL was probably related to surface trap effect.23 HRTEM imaging of APTES-ZnO QDs indicated a particle size of 7 ± 2.1 nm and a very thin silane coating as it was not observed anymore (Figure 1d).23,24 The ζ potential was +11.2 ± 0.37 mV (Figure 1e). The free amino groups of APTES are the origin of this positive ζ potential value, and they can impart good dispersion and stability, especially in water.25

2.2. Synthesis of MgAl-Cl-LDH (Pristine LDH)

MgAl-Cl-LDH NPs were successfully prepared by coprecipitation of mixed magnesium and aluminum salts (MgCl2·6H2O and AlCl3·6H2O) solution in an alkaline NaOH solution, under nitrogen, followed by hydrothermal treatment to improve particle size distribution and crystallinity as shown in Figure 2. The molar fraction of Al3+ in the prepared L1 and F1 was 0.33 that matches with the upper limit reported in the literature (0.2 ≤ x ≤ 0.33).26 The stoichiometric ratio (MgCl2/AlCl3) was (2:1) (Table S2). The high charge density exhibited by this stoichiometric molar ratio increased the gallery height of LDH brucite due to electrical repulsion, thus probably allowing the easy intercalation of the guest anion.27 Magnesium and aluminum elemental analysis of F1 by inductively coupled plasma-optical emission spectrometry (ICP-OES) confirmed Mg2+/Al3+ molar ratio (Table S1).

Figure 2.

Figure 2

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Schematic representation of the synthesis of APTES-ZnO QDs conjugated to sericin-modified LDH-PMX.

2.3. Synthesis of Pemetrexed Intercalated MgAL-Cl-LDH (LDH-PMX)

The intercalation of PMX, a dicarboxylic acid drug, into the LDH was illustrated in Figure 2. Several attributes such as exchange media pH, anion concentration, ionic radius of guest anion, and chemical composition of LDH (charge density and hydration state) usually affect the intercalation of anionic drugs into the brucite layers of LDH via anion exchange.28,29 Therefore, in this study, the pH was kept at 9 to ensure that PMX was entirely ionized, thus allowing easy intercalation into the positive LDH layers. Besides, the amount of guest anion is crucial for intercalation into LDH galleries by anion exchange route rather than adsorbed on the LDH surfaces. Generally, the isomorphic substitution of Mg2+ by Al3+ during LDH precipitation leads to positive charge increase and is usually balanced by the interlayer anions as Cl–. Consequently, the amount of Al3+ can be considered as a rough estimate of the interlayer balancing anion amount.30 Moreover, the amount of guest anion should be around double that of the existing interlayer anion (e.g., Cl– and NO3–) to ensure efficient guest anion intercalation through the anion exchange process.31 In this study, we examined the intercalation of a certain amount of PMX into the previously prepared LDHs; L1 and F1 which resulted in the formation of L2 and F2, respectively. The amount of PMX relative to Al3+ in F2 is double that in L2, which enhanced PMX intercalation within the LDH brucite layers in F2 (Table S2). Furthermore, F2 showed better particle size distribution and higher drug loading (DL) (5 ± 0.45 and 10.7 ± 0.62% for L2 and F2, respectively) (Table S2).

2.4. Modification of LDH2-PMX with Sericin (Seri@LDH2-PMX)

As shown in Figure 2, the desolvation technique was employed to entrap F2 within sericin.14,15F2: sericin weight ratio of 0.1:1, 0.2:1, 0.3:1, 0.4:1, or 1:1 was investigated. Genipin (GNP) 10% (w/w) was employed as a crosslinker with several F2: sericin weight ratios following the desolvation technique. The particle size seems to increase with the increase in F2 weight relative to sericin to reach 315.1 ± 2.6 nm at 0.3:1 weight ratio with a polydispersity index (PDI) of 0.35 ± 0.03, followed by a decrease in particle size at 0.4:1 weight ratio (200.62 ± 0.9 nm), in addition to a decrease in PDI (0.204 ± 0.04). The optimized Seri@LDH2-PMX (GNP) (F3) of 1:1 weight ratio showed a slight improvement in PDI (0.18 ± 0.03) compared to the 0.4:1 ratio (Figure 3a). The ζ potential was insignificantly (P-value > 0.05) increased from −31.6 ± 1.8 to −20.8 ± 0.3 mV, where more positively charged F2 NPs were added into the system (Figure 3b).

Figure 3.

Figure 3

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Effect of weight ratios of LDH2-PMX (F2): sericin crosslinked with genipin on (a) particle size distribution, PDI, and (b) ζ potential. Effect of glutaraldehyde (GTA) amount on (c) particle size, PDI, and (d) ζ potential of the desolvated nanohybrids.

On the other hand, several volumes of 8% (v/v) GTA were examined for crosslinking of 1:1 weight ratio of F2: sericin following the desolvation technique. The particle size and PDI significantly decreased (P-value < 0.05) with the decrease in GTA amount. The smallest particle size (175.7 ± 0.022 nm) was obtained in the presence of 35 μL 8% (v/v) GTA to afford the optimized F4 formula, showing an improved PDI (Figure 3c). This could be attributed to the GTA interconnections between sericin macromolecules, that hold them together, leading to larger particle size and PDI value. Actually, GTA reacts immediately with the accessible lysine residues with possible steric hindrance and self-polymerization to give GTA oligomers of high molecular mass, so it is necessary to add the bifunctional reagent (GTA) gradually and in small amounts.32 On the other hand, the ζ potential insignificantly decreased (P-value > 0.05) with increasing GTA concentrations (Figure 3d). All ζ potential measurements ranged from −13.9 ± 0.23 to −24.1 ± 0.4 mV. The contribution of the negatively charged carboxylic groups increased when positively charged amino groups are crosslinked, resulting in a drop in ζ potential.

2.5. Preparation of APTES-ZnO QDs Conjugated to Sericin-Modified LDH-PMX (APTES-ZnO QDs/Seri@LDH2-PMX)

APTES-ZnO QDs were chemically conjugated to sericin protein by carbodiimide coupling reaction, as shown in Scheme 1.33−36 The coupling reagents EDC·HCl/K·Oxyma preactivated the carboxylic acid groups of sericin by forming an intermediate active ester liable for covalent bonding through nucleophilic substitution by the amino groups of APTES-ZnO QDs. Different sericin/APTES-ZnO QDs molar ratios (1:1, 1:2, and 1:3) were investigated for the luminescence properties of the QDs after chemical conjugation. The molar ratio 1:3 was selected in this study where the PL of the QDs was preserved and static quenching was prevented compared to other molar ratios (Figure 6c).

Scheme 1. Chemical Conjugation (Carbodiimide Coupling) of APTES-ZnO QDs with Sericin Protein.

Scheme 1

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Figure 6.

Figure 6

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(a) Fourier transform infrared (FT-IR) spectra of LDH2 (F1), LDH2-PMX (F2), APTES-ZnO QDs/Seri@LDH2-PMX (GTA) (F6), and sericin. (b) Differential scanning calorimetry (DSC) thermograms of LDH2 (F1), LDH2-PMX (F2), APTES-ZnO QDs/Seri@LDH2-PMX (GTA) (F6), and sericin.

Accordingly, APTES-ZnO QDs were chemically conjugated to F3 and F4 based on the optimum molar ratio yielding the final formulations; F5 and F6, respectively (Figure 2). The GTA crosslinker in F6 did not interfere with the PL of APTES-ZnO QDs as detailed in Section 2.6.5, which proved that GTA is more suitable crosslinker than GNP in F5. Accordingly, F6 formula was chosen to be the ideal nanotheranostics in our study.

2.6. Physiochemical Characterization of APTES-ZnO QDs/Seri@LDH-PMX

2.6.1. X-ray Powder Diffraction (XRD)

X-ray photoelectron spectroscopy (XPS) and other methods are frequently used to examine the surface chemistry of a material. Metals’ elemental composition, empirical formula, chemical state, and electronic state can all be determined. Its use in the characterization of organic molecules (such PMX and sericin) is, however, very restricted. In this study, we employed the XRD technique for the qualitative crystallinity investigation. High crystallinity might lead to the creation of a microchannel structure, whereas at the same time, the polymer matrix’s vast surface area might enhance the drug release from the NPs.37 XRD plots of L1 and F1 represent the structural formula Mg6Al2(OH)18·4.5H2O, registered by the standard card 35-0965 of the Joint Committee on Powder Diffraction Standards (JCPDS), and the standard model of the R3̅m [166] crystal system with the lattice parameters of a = b = 0.305400 and c = 2.340000. Their well-crystallized layered structure was confirmed by different crystal planes as shown in Figure 4a. In accordance with Bragg’s equation, nλ = 2d sin θ,17 the d-spacing was determined, as n is the reflection order and usually equals 1, λ is the X-ray wavelength (1.5406 Å), d is the d-spacing, and θ is the incident X-ray angle in radians. Table 1 reveals that L1 and F1 have a basal spacing (d-spacing) of 7.8 Å which is calculated from (003) plane reflection with a brucite layer thickness of 3.89 Å, this is in agreement with the literature.38 Accordingly, the gallery height (interlayer spacing) is 3.91 Å (Figure 4a). Additionally, Scherrer’s formula; D = Kλ/β cos θ,39 was used to calculate the crystallite sizes (Table 1). The crystallite size (D) is in Å, Scherrer’s constant (K) equals 0.9, the X-ray wavelength (λ) is 1.5406 Å, Bragg’s angle (θ) is in radians, and the full width at half-maximum (FWHM) β is in radians and describes how broad the peak is.

Figure 4.

Figure 4

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(a) XRD patterns of LDH1 (L1), LDH1-PMX (L2), LDH2 (F1), LDH2-PMX (F2), and APTES-ZnO QDs/Seri@LDH2-PMX (GTA) (F6); (b) schematic diagram showing the intercalation of PMX into LDH2 (F1) to obtain LDH2-PMX (F2); TEM images of (c) LDH2 (F1), (d) LDH2-PMX (F2), and (e) APTES-ZnO QDs/Seri@LDH2-PMX (GTA) (F6); and (f) SEM image of LDH2 (F1).

Table 1. XRD Data of LDH1 (L1), LDH2 (F1), and LDH2-PMX (F2) in (Å).
plane 2θ (deg) d-spacing (Å) FWHM crystallite size (Å)
XRD data of L1 and F1 in (Å)
003 11.335 7.800087 1.13197 7.052584
006 22.842 3.890078 1.25225 6.472170
012 34.742 2.580070 1.63366 5.095265
015 39.133 2.300085 3.38598 2.490020
118 46.358 1.957038 4.59879 1.879160
110 60.588 1.527048 8.83270 1.041658
113 61.889 1.498033 8.41200 1.101128
XRD data of F2 in (Å)
003 10.83490 8.158962 0.381120 20.93811
006 22.57079 3.936204 0.485430 16.68814
012 34.36897 2.607215 28.61365 0.290613
015 38.47922 2.337647 24.06545 0.349640
118 45.84467 1.977750 38.52123 0.223913
110 60.04183 1.539627 5.640800 1.626579
113 61.38039 1.509221 6.678300 1.383315
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XRD pattern shown in Figure 4a indicated that the intercalation of PMX into L1; L2 produced a significant change in the crystalline structure of L2 as the (003) diffraction peak disappeared. Subsequently, PMX is suggested to be adsorbed or simply bound to the LDH surfaces in L2 rather than intercalated within the brucite layers, this may be attributed to the low concentration of PMX relative to Al3+ in LDH as discussed earlier.31,40 Conversely, the intercalation of a higher concentration of PMX relative to Al3+; F2 showed a typical XRD pattern as the layered structure of F1 with its characteristic diffraction peaks (Figure 4a). Compared to F1 XRD plot, the (003) and (006) diffraction peaks of F2 are lower intensity and much broader, indicating that the crystallinity of the LDH phase is reduced because of PMX intercalation. Furthermore, because the peak corresponding to the (003) plane was displaced from 11.335 Å in F1 to a lower angle of 10.835 Å in F2, the d-spacing for the basal plane (003) was slightly increased from 7.8 Å in F1 to 8.16 Å in F2 (Table 1). The nonsignificant increase of the interlayer spacing after PMX intercalation may suggest the horizontal pattern of intercalation rather than the vertical one.31,41 From the above findings, F1 was selected in our study. Figure 4b illustrates the intercalation of PMX into F1 producing F2. As reported, the amorphicity of sericin is revealed by a broad peak at 2θ value of approximately 20° in the XRD pattern.42 XRD pattern of F6 (Figure 4a) shows a similar amorphous pattern as sericin, suggesting that LDH surfaces are well modified with sericin protein.43 It should be noted that the average distance between the metal ions and Mg2+/Al3+ ratio can be assessed through the value of lattice parameter a calculated from (110) diffraction (2θ = 60°).44 The prepared formulations convey approximately the same value a as shown in the XRD plots (Figure 4a), indicating a nonsignificant Mg2+/Al3+ ratio change.

2.6.2. Particle Size Distribution, ζ Potential, and Intercalation Efficiency

L1 particle size (119 ± 0.021 nm) was markedly increased compared to F1 (131.8 ± 0.03 nm) after PMX loading, producing L2 (532.8 ± 1.23 nm) and F2 (136.3 ± 0.59 nm), respectively (Table 2). The ζ potential of L1 was inverted from highly positive +43.2 ± 0.4 mV to negative −12 ± 0.93 mV for L2. On the other hand, the ζ potential showed a slight decrease from +41.8 ± 0.36 mV for F1 to +34.8 ± 0.27 mV for F2 (Table 2). These findings are consistent with the PMX surface adsorption possibility in L2 and the PMX intercalation in F2 which were suggested previously by XRD analysis. As discussed previously, 1:1 weight ratio of F2: sericin using 10% (w/w) GNP as crosslinker was selected to prepare F3 based on the particle size and PDI analysis (Figure 3a). Comparatively, 35 μL of 8% (v/v) GTA offered the best particle size and PDI for 1:1 weight ratio of F2: sericin in the preparation of F4 (Figure 3c). Moreover, F5 particle size was 218.5 ± 0.21 nm with a slight decrease in ζ potential (−18.4 ± 0.25 mV) compared to F3 owing to the partial utilization of carboxylic groups of sericin (amino groups will predominate) by the chemical conjugation to the QDs amino groups (Table 2). F6 showed a particle size of 201.9 ± 2.3 nm and a slight decrease in ζ potential (−21.1 ± 0.51 nm) compared to F4 (Table 2). It should be noted that the ζ potential values of the prepared nanocarriers were measured in neutral pH to mimic the blood conditions after IV injection, to maintain the stability of NPs, and to prevent the premature release of PMX (i.e., PMX release in the blood rather than at the tumor site, as both LDH and sericin are sensitive to acidic pH). Intercalation efficiencies (IE %) of all prepared formulations are presented in Table 2.

Table 2. Composition, Physicochemical Characteristics, Particle Size, ζ Potential, and Intercalation Efficiency (IE) and Drug Loading (DL) of Blank and Drug Loaded NPs (n = 3).
  formulation PMX (g) particle size (nm) ζ potential (mV) PDI PMX IE % PMX DL %
L1 LDH1   119.0 ± 0.02 +43.2 ± 0.4 0.167 ± 0.02    
F1 LDH2   131.8 ± 0.03 +41.8 ± 0.36 0.152 ± 0.01    
L2 LDH1-PMX 0.03 532.8 ± 1.23 –12 ± 0.930 0.246 ± 0.04 99.64 ± 0.3 5 ± 0.45
F2 LDH2-PMX 0.03 136.3 ± 0.59 +34.8 ± 0.27 0.304 ± 0.01 99.40 ± 1.1 10.7 ± 0.62
F3 Seri@LDH2-PMX (GNP) 0.03 200.6 ± 0.90 –20.8 ± 0.11 0.204 ± 0.03 95.13 ± 0.5 8.6 ± 0.51
F4 Seri@LDH2-PMX (GTA) 0.03 175.7 ± 0.022 –24 ± 0.730 0.221 ± 0.01 93.16 ± 1.3 8.18 ± 0.51
F5 APTES-ZnO QDs/Seri@LDH2-PMX (GNP) 0.03 218.5 ± 0.21 –18.4 ± 0.25 0.303 ± 0.04 92.80 ± 0.94 6.56 ± 0.22
F6 APTES-ZnO QDs/Seri@LDH2-PMX (GTA) 0.03 201.9 ± 2.30 –21.1 ± 0.51 0.294 ± 0.02 90.70 ± 0.65 6.46 ± 0.32
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2.6.3. Morphological Analysis, Microscopy, and Surface Topography

Upon TEM examination, F1 NPs were almost hexagonal plate-like particles, endorsing hydrotalcite typical features, and having a smooth surface. The measured particle size was about 111 nm (Figure 4c). Moreover, the morphology of F2 remained unchanged following PMX loading (Figure 4d). The measured size of F2 by TEM was around 131.2 nm. Conversely, the modification of LDH2 with sericin in the final formula F6 produced many irregularly shaped nanometer crystal particles which were found to stack together (Figure 4e). Compared to the size measured by dynamic light scattering (DLS), it is obvious that the apparent size measured by TEM is much less, as throughout preparation for TEM examination, particle dehydration induces shrinkage.

The high-magnified images of F1 morphology and surface topography obtained using SEM revealed a great number of hexagonal platelets with lamellar structure shaped with a very narrow nanosized range and had smooth surfaces. They are uniformly distributed all over the sample (Figure 4f).

2.6.4. UV Absorption

Absorption spectra were recorded to confirm the crosslinking reaction after sericin desolvation. Two absorption peaks appeared at ∼280 and ∼600 nm in both F3 and F5 (Figure 5a). The crosslinked products, obtained by the reaction of primary amino groups of biopolymers with GNP, have been reported to afford light absorption in different regions of light.45 By increasing the crosslinking time using GNP, the reaction color of F3 and F5 gradually changed from yellow to green, then to light blue, and eventually to dark blue.46 The large π–π* conjugation system of the GNP-crosslinked products is responsible for this blue fluorogenic characteristic, thereby confirming Schiff’s bases generation between the amines of sericin and GNP.47 Although GTA-induced interlayer sericin crosslinking does not have a significant color change, it is more likely to react with the amino groups to form Schiff’s bases than GNP.47 Therefore, it is expected that the interlayers of sericin protein in F4 and F6 were crosslinked via Schiff’s bases.

Figure 5.

Figure 5

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(a) UV absorption spectra of Seri@LDH2-PMX (GNP) (F3) and APTES-ZnO QDs/Seri@LDH2-PMX (GNP) (F5); (b) photoluminescence (PL) spectrum of sericin upon excitation at λ = 330 nm; (c) effect of different sericin/ZnO QDs molar ratios on the photoluminescence (PL) of APTES-ZnO QDs upon excitation at λ = 330 nm; (d) photoluminescence (PL) spectra of Seri@LDH2-PMX (GNP) (F3) and APTES-ZnO QDs Seri@LDH2-PMX (GNP) (F5) compared with APTES-ZnO QDs; and (e) photoluminescence (PL) spectra of APTES-ZnO QDs/Seri@LDH2-PMX (GTA) (F6) and ZnO QDs-sericin conjugate C compared with APTES-ZnO QDs.

2.6.5. Photoluminescence (PL)

In principle, it was reported that sericin could intrinsically emit fluorescence when excited by light.48 Meanwhile, the emission spectrum of sericin solution upon excitation at 330 nm shows a relatively low-intensity peak at 433 nm (Figure 5b). The intrinsic protein fluorescence is reported to be produced by the aromatic amino acids particularly tyrosine, phenylalanine, and tryptophan.49 Indeed, the quantum yields of these naturally occurring fluorophores are relatively low and, accordingly, their brightness. In addition, their highest emission peak intensity usually lies in the UV region of light. That makes them less suited for fluorescence microscopy as it is generally optimized for bright fluorophores which emit in the visible region of light (380–740 nm).49 Herein, extrinsic fluorophores, particularly QDs, are more suitable for bioimaging purposes. QDs are characterized by several optical properties which urge their use as fluorescent probes including both NIR and visible region emitting ability.3 Emission spectra of sericin/APTES-ZnO QDs conjugates with different molar ratios were investigated as shown in Figure 5c; sericin–ZnO QDs conjugates A, B, and C with sericin/ZnO QDs molar ratios 1:1, 1:2, and 1:3, respectively. It is clear that sericin–ZnO QDs conjugates A and B do not show emission peaks at the region of ZnO QDs emission (∼533 nm). However, their emission peaks were transferred to sericin emission region (∼433 nm). Upon increasing the QDs concentration, 1:3 molar ratio (sericin–ZnO QDs conjugate C), the PL was shifted into the QDs region (∼533 nm). As observed, the covalent interaction between QDs and sericin at low concentrations of QDs leads to charge transfer from QD to sericin, therefore, quenching the fluorescence of QDs. Generally, static quenching (complex formation), dynamic quenching (collisional quenching), excited state reactions, and energy transfer are all known to cause fluorescence quenching.50 Static quenching, which is known for its concentration-dependent manner, is supposed to be the quenching mechanism resulting from the covalent interaction that leads to a complex formation between QDs and sericin. Since 1:3 molar ratio of sericin/APTES-ZnO QDs attains the most suitable PL properties, it was selected. Unfortunately, QDs fluorescence quenching in F5 was observed (Figure 5d). The detected emission peaks in both F3 and F5 are owing to GNP-crosslinked products which are known to fluoresce at 380–700 nm (Figure 5d).45 At ∼422 nm, the small peak is attributed to Raman scattering of H2O solvent,51 besides, a broad emission peak at ∼470 nm45 (Figure 5d). As previously discussed in Section 2.6.4, F5 can absorb light at ∼600 nm which is nearby the emission region of APTES-ZnO QDs (∼533 nm) (Figure 5a). Therefore, the emitted QDs fluorescence can be absorbed by F5, leading to a quenching process that may be justified by the inner-filter effect. The absorption of exciting light, leaving a low-intensity luminous flux reaching the fluorescent material is known as the primary inner-filter effect. Compared to this, the secondary inner-filter effect occurs due to reabsorption of fluorescence.52 This quenching process greatly complicates the measurement of fluorescence emission as the concentration of the fluorescent substance is nonlinearly dependent on its fluorescence intensity.53 On the other hand, the PL of APTES-ZnO QDs was preserved in F6 (Figure 5e). These results indicated that GTA is a more suitable crosslinker than GNP, thus F6 would be our ideal optimized nanotheragnostics.

2.6.6. Fourier Transform Infrared (FT-IR) Spectroscopy

FT-IR spectrum of F2 (Figure 6a) revealed peaks corresponding to PMX in the range of 3600–2600 and 1660 cm–1 corresponding to the O–H stretching vibration (carboxylic acid) and C=O amide, respectively, indicating the interaction of PMX into LDH. FT-IR spectrum of F6 (Figure 6a) shows bands at 3267, 1661, and 1554 cm–1 corresponding to N–H amide A, C=O amide I, and C=O amide II, respectively, corresponding to sericin.54 The peak located at 1357 cm–1 could be due to carbonate contamination in LDH. Besides, peaks located at 789, 683, and 555 cm–1 are due to lattice vibration and bending of LDH.55 On the other hand, the broad band in the range of 3500–3100 cm–1 corresponds to the O–H stretching vibration (carboxylic acid) of PMX (Figure 6a).

2.6.7. Differential Scanning Calorimetry (DSC)

The thermogram of LDH2 (F1) shows an endothermic peak at 108.6 °C which could be associated with the removal of adsorbed moisture. Besides, two other endothermic peaks were observed at 239 and 364 °C which could be attributed to the loss of water and charge-balancing interlayer ions from the interlayer space.56 In addition, an exothermic peak is observed at 440 °C (Figure 6b). PMX is reported to acquire three characteristic endothermic peaks at 91.8, 153.8, and 243.8 °C.25 LDH loaded pemetrexed, LDH2-PMX (F2) thermogram showed three endothermic peaks at 99.9, 200, and 390 °C in addition to an exothermic peak at 320 °C. The observable shift in PMX decomposition temperature in F2 from 91.8 to 99.9 °C can be attributed to PMX increased thermal stability in the interlayer space of LDH in comparison with its free state (Figure 6b). The DSC thermogram of sericin showed the first endothermic peak at a range of 25–100 °C with a maximum of 88.4 °C that could be related to the evaporation of adsorbed water from the protein. Two endothermic peaks were observed at 228 and 388 °C which could be associated with progressive deamination, decarboxylation, and depolymerization as a result of the breakdown of (poly)peptide bonds. In addition, an exothermic peak is observed at 468 °C corresponding to sericin primary structure carbonization (Figure 6b).57 APTES-ZnO QDs/Seri@LDH2-PMX (GTA) (F6) thermogram shows one broad endothermic peak with a maximum of 156 °C. In addition, two broad exothermic peaks were observed at 310 and 440 °C (Figure 6b). On the other hand, the endothermic peaks of both sericin and PMX drug disappeared. These results could confirm the conversion of PMX to its amorphous state within the fabricated F6 NPs.

2.7. In Vitro Drug Release

Drug release profiles were determined in vitro for F2, and F6 compared to free PMX. As shown in Figure 7a, the free PMX was completely (100%) released after almost 2 h at both pH 7.4 and 4.8. However, a gradual and biphasic release profile of PMX is demonstrated with F2, starting with an early fast release (27.8 and 48.5% after 0.5 h at pH 7.4 and 4.8, respectively), followed by a much slower release that eventually results in the release of the whole PMX (98.34% after 7 h and 99.8% after 5 h at pH 7.4 and 4.8, respectively). The initial burst release of PMX observed in the case of F2 could be due to the release of the drug adsorbed on LDH surfaces. As noted, the release time of F2 is shorter at pH 4.8 than at pH 7.4, which could be justified by the difference in PMX release mechanism at different pHs. In detail, at pH 7.4, only an anion exchange mechanism with PO3– in the release media allowed the drug molecules to be released. Nevertheless, two processes; anion exchange and dissolution have been stated in the acidic medium (pH 4.8). As LDH hydroxyl groups become protonated at low pH, the LDH lattice undergoes degradation, thus enhancing the drug release.58 Analogously, F6 showed a biphasic release modality. After the first 2 h about 19.2 and 44.3% were released at pH 7.4 and 4.8, respectively. Then, a much slower release of about 99.23% after 30 h and 99.3% after 11 h at pH 7.4 and 4.8, respectively. It is obvious that the acidic pH showed a faster release profile, as the carboxylic groups of aspartic acids of sericin become protonated; hence, the electrostatic repulsion between NPs decreased, causing them to swell and enhancing the PMX release rate from F6. Similarly, doxorubicin pH-dependent release behavior from silk protein NPs was reported.59 This pattern of drug release reveals the superior stability of F6 in physiological pH over the tumor acidic pH gaining a more efficient drug delivery. It should be noted that the difference in the release rate of PMX between F2 and F6 was attributed to the sericin coating of LDH in F6 which has a stabilizing effect, leading to the reduction of the amount of PMX released.55 Slow and sustained release of a drug is reported to gain a more efficient drug delivery with fewer side effects.60 Furthermore, the biphasic and sustained release profile provided by this formula; F6 may be therapeutically beneficial because the initial rapid release allows the therapeutic dose effect, and the later sustained pattern maintains that dose. It should be noted that we do not consider the ζ potential measurement of the formulations at different release pH. As the ζ potential is highly influenced by pH, it is expected that the ζ potential will be more positive at low pH and more negative at high pH.

Figure 7.

Figure 7

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(a) In vitro release study of free PMX, APTES-ZnO QDs/Seri@LDH2-PMX (GTA) (F6), and LDH2-PMX (F2) at pH 7.4 and 4.8 at 37°C; (b) hemolytic potential of APTES-ZnO QDs/Seri@LDH2-PMX (GTA) (F6) and LDH2-PMX (F2) showing % hemolysis of different concentrations; (c) in vitro hemolysis test images after 1 h incubation period at 37 °C for APTES-ZnO QDs/Seri@LDH2-PMX (GTA) (F6) and LDH2-PMX (F2); and (d) in vitro serum stability of APTES-ZnO QDs/Seri@LDH2-PMX (GTA) (F6) and LDH2-PMX (F2) with 10% (v/v) fetal bovine serum (FBS) according to particle size and PDI measurements at different time intervals using DLS.

2.8. In Vitro Hemolytic Test

Throughout the development of parenteral nanomolecular systems, hemocompatibility is known to be a significant issue. Different concentrations in the range of 0.5–2 mg/mL were examined for hemocompatibility.61 The amount of hemoglobin released from RBCs by F6 was compared with F2. The minimum hemolysis (1.51 ± 0.1%) was produced when using F6 at a quantity of 0.5 mg/mL, and 3.68 ± 0.32% when using a quantity of 2 mg/mL (Figure 7b). Those amounts are far less than 5%, the acknowledged harmless limit. The hemolytic activity of F6 is minimal in the concentration range evaluated in this study, indicating that it can be safely administered intravenously. On the other hand, F2 showed an obvious hemolytic activity starting with 6.48 ± 0.21% at the lowest concentration tested, 0.25 mg/mL, with progressive hemolysis increasing with increasing F2 concentration to reach 36.6 ± 0.1% at 2 mg/mL (Figure 7b). The negatively charged RBCs could easily interact with the positively charged F2 causing RBC disruption and subsequently hemoglobin release.62 It should be noted that the reported hemocompatibility of LDH could be related to the low concentrations of LDH employed in the hemolytic study compared with the concentrations tested in this study.63 It is obvious that sericin increased the hemocompatibility of LDH which could be attributed to the negative charges imparted by sericin in addition to colloidal stability enhancement, thus keeping RBC integrity by the generated repulsion forces. In vitro hemolytic test images after 1 h incubation period at 37 °C for F6 and F2 are presented in Figure 7c.

2.9. In Vitro Serum Stability

Inorganic NPs, particularly LDH, suffer from colloidal instability and undergo aggregation because of pH or ionic strength change. An earlier observation by Gu et al. suggested that the electrostatic precoating of LDH with bovine serum albumin (BSA) elicits higher cellular uptake efficiency compared with uncoated LDH. This could be attributed to the smaller size resulting from enhanced colloidal stability by BSA precoating.64 In our study, the particle size of F6 did not show a significant change (P-value > 0.05) with zero-time addition of fetal bovine serum (FBS) (205.7 ± 0.8 nm) compared with the original particle size (201.9 ± 2.3 nm). After 5 h, the particle size reached 268.9 ± 0.24 nm and decreased to 259 ± 0.8 nm after 6 h (Figure 7d). Protein molecules are sometimes associated with the surfaces of NPs and then dissociated, explaining this fluctuation in the particle size.65 Also, the small change in PDI of F6 from 0.25 to 0.391 during the entire experiment indicated its high stability. The repulsive forces generated between the negatively charged serum proteins and the sericin protein in F6 could explain the high serum stability. Considering therapeutic applications, neutral or negatively charged nanomolecular systems are generally more favorable than positively charged ones. Conversely, upon adding FBS (0 time) to F2, the particle size showed a significant (P-value < 0.05) increase from 136.2 ± 0.59 to 419.8 ± 2.5 nm, indicating their colloidal instability. The positive surface charge of F2 NPs is thought to enhance the adsorption of negatively charged serum proteins, resulting in their aggregation.

2.10. Physical Stability

Based on particle size and ζ potential change over time, the physical stability of APTES-ZnO QDs/Seri@LDH2-PMX (GTA) (F6) was assessed after 3 months of storage at 4 °C (Table S6). Following 3 months of storage, the formula showed a particle size of 287.2 ± 2.71 nm compared with the original particle size (201.9 ± 2.3 nm). In addition, the ζ potential values remained nearly unchanged (−20.9 ± 0.63 mV) compared with the original ζ potential (−21.1 ± 0.51 mV), which reflects its stability (Table S6).

2.11. Freeze Drying and Redispersibility

Lyophilization (freeze drying) has long been considered to control the long-term stability of nanosystems. F6 retained its original physicochemical properties as it demonstrated good aqueous redispersibility after freeze drying, indicating that it could be robust for pharmaceutical applications. The reconstituted F6 had a particle size of 215.7 ± 1.2 nm and a redispersibility index (RI) of 1.067 in this study (Table S7). A range of 0.95–1.07 for RI has been found to be typically acceptable.66 In addition, freeze drying did not cause a significant ζ potential change (Table S7). This implies the nanocarriers’ stability with long-term storage as a lyophilized powder.

2.12. In Vitro Cytotoxicity Study and Cellular Uptake

2.12.1. In Vitro Cytotoxicity Study

Cell viability testing of free APTES-ZnO QDs, free PMX, LDH2 (F1), and APTES-ZnO QDs Seri@LDH2-PMX (GTA) (F6) was carried out on MDA-MB-231 cells at 24 and 72 h (Figure 8). Blank NPs; F1 did not have a significant inhibitory effect on cell viability at low concentrations up to 6.25 μg/mL. However, at higher concentrations, it exhibited inhibitory effects on the cell viability, manifested by a reduction in cellular viability to reach 57.8% after 72 h treatment with a concentration of 100 μg/mL. LDH NPs are reported to be more cytotoxic on cancer cells, compared to normal cells. A study by Choi et al. reported higher cytotoxicity of LDH which was manifested with reactive oxygen species (ROS) generation, lactate dehydrogenase leakage, and IL-8 release on A549 lung cancer cells, compared with normal L-132 cells exposed to equimolar concentrations.18 APTES-ZnO QDs were employed in the current study mainly for bioimaging purposes. They did not show an inhibitory effect at low concentrations (0.39, 1.59 μg/mL); however, they were found to exert some inhibitory effect on cell viability at higher concentrations retaining 51.4% of the cell viability after 72 h exposure to a concentration of 100 μg/mL (Figure 8). ZnO QDs are dispersed and small in size compared to other ZnO nanoforms, and are therefore expected to greatly increase cellular uptake and have a higher cytotoxic potential.67 ZnO QDs are attracted to the acidic tumor microenvironment, which causes them to decompose and release Zn2+ thus enhancing targeted therapy without affecting the normal cells. It should be noted that ZnO QDs were attached via an amide bond, which is a relatively stable bond under various conditions. Thus, it takes a longer time to be cleaved under the effect of enzymatic reactions allowing cellular imaging before ZnO degradation in the acidic tumor environment.68 Moreover, ZnO QDs are reported to induce cell cycle arrest at the G0/G1 phase of MCF-7 and MDA-MB-231 cells, thereby inducing their apoptosis.67 On the other hand, APTES-ZnO QDs Seri@LDH2-PMX (GTA), F6 showed a greater inhibitory effect on cell viability at all of the concentrations tested compared to free APTES-ZnO QDs, LDH2 (F1) and free PMX at equivalent concentrations (Table S10). The cytotoxic effect of F6 was time- and concentration-dependent (Figure 8). The IC50 values of F6 were 1.34 ± 0.03 and 0.31 ± 0.007 μg/mL at 24 and 72 h, respectively which were significantly lower than that of all investigated formulations (Table 3). The increased of cytotoxicity of F6 compared with free PMX (about 60-fold reduction of IC50 compared to free PMX after 72 h) could be attributed to the higher uptake and efficient intracellular retention. Sericin, like other proteins, is likely to enhance the cellular uptake of inorganic NPs. As reported earlier, the precoating of LDH with albumin demonstrated higher cellular uptake efficiency compared with uncoated LDH.64,70 Cellular internalization and conversion into penta-polyglutamated form are the key prerequisites for the demonstrated high cytotoxicity effect of PMX.20 Accordingly, we can conclude that our proposed system enhanced the efficacy of PMX at the cellular level. Within this engineered system, PMX is maintained and protected in the interlayer LDH, so it is anticipated to reach the tumor without being released in the blood, allowing it to accumulate within the tumor cell. Also, it can bypass cellular resistance mechanisms such as P-glycoproteins that can efflux various anticancer drugs out of cancer cells. Moreover, the modification of LDH with sericin, like other natural polymers, can provide longer blood circulation by preventing phagocytosis and opsonization, thus attaining good immune escape properties and enhancing the LDH biosafety eventually.71,72

Figure 8.

Figure 8

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Cell viability of free APTES-ZnO QDs, free PMX, LDH2 (F1), and APTES-ZnO QDs/Seri@LDH2-PMX (GTA) (F6) on MDA-MB-231 breast cancer cells.

Table 3. IC50 of Free APTES-ZnO QDs, Free PMX, F1, and F6 on MDA-MB-231 Breast Cancer Cells at 24 and 72 h.
  IC50 (μg/mL)
  MDA-MB-231 cells
compound 24 h 72 h
free APTES-ZnO QDs 243.24 ± 5.91 199.06 ± 4.84
free PMX 33.49 ± 0.81 23.34 ± 1.08
pristine LDH2 F1 570.40 ± 13.87 415.26 ± 11.38
APTES-ZnO QDs/Seri@LDH2-PMX (GTA) F6 1.34 ± 0.03 0.31 ± 0.007
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2.12.2. In Vitro Cellular Uptake

2.12.2.1. Confocal Microscopy Study

Fluorescent images were obtained by confocal laser microscopy (CLSM) and used to analyze the cellular uptake efficiency of F6, relative to free APTES-ZnO QDs, by MDA-MB-231 breast cancer cells (Figure 9a). A relatively low intensity of green fluorescence was observed in MDA-MB-231 cells treated with free QDs after 24 h, indicating their low cellular uptake. Comparatively, a higher intensity of green fluorescence was noticed after 24 h incubation with F6, indicating their higher cellular uptake compared with free QDs (Figure 9b). According to the literature, ZnO QDs with green and yellow emissions (520–550 nm) are suitable for biological labeling compared with blue emission because most cells and tissues can also absorb light in this region.73 As observed, the current work ended with in vitro cellular uptake which confirmed cellular labeling and bioimaging efficiency of the prepared formulation. For future in vivo studies, the upconversion luminescence may be employed in which the ZnO QDs will be irradiated at a longer wavelength (to minimize tissue absorption) while keeping the emission at a shorter wavelength.74

Figure 9.

Figure 9

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Confocal images of MDA-MB-231 breast cancer cell line treated with (a) free APTES-ZnO QDs and (b) APTES-ZnO QDs/Seri@LDH2-PMX (GTA) (F6) after 4 and 24 h.

The physio-pathological features of tumor vasculature, as being highly disordered and having multiple fenestrations, are known to passively permit nanocarriers, especially those in the 100–200 nm size range, to seep through tumor blood vessels and accumulate in cancer cells via EPR effect.75 The sericin obtained from B. mori is known to hold several mannose residues with its oligosaccharide units.76 These mannose residues may interact with overexpressed mannose receptors on cancer cell surfaces, increasing cellular uptake through active tumor targeting.25 Moreover, we observed in this study that the colloidal stability of LDH was enhanced after the modification with sericin protein that may also explain the efficient cellular uptake as previously reported for LDH coated with BSA.64

2.12.2.2. Flow Cytometry Study

As shown in Figure 10a, flow cytometry was utilized for quantitative analysis of the intracellular uptake efficiency of F6 and free APTES-ZnO QDs. The mean fluorescence intensity (MFI) describes cellular internalization (Figure 10b). After 24 h, there was a higher uptake efficiency of F6 than after 4 h (P-value < 0.05) (Figure 10b). The CLSM images and cytotoxicity data corroborate these findings.

Figure 10.

Figure 10

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(a) Flow cytometry analysis of MDA-MB-231 breast cancer cell line treated with APTES-ZnO QDs/Seri@LDH2-PMX (GTA) (F6), and free APTES-ZnO QDs for 4 and 24 h 37 °C. (b) Flow cytometry results in form of MFI.

3. Experimental Section

3.1. Materials and Methods

The details of materials including drugs, chemicals, kits, and solvents are described in the Supporting Information (SI).

3.2. Preparation of ZnO Capped by APTES QDs (APTES-ZnO QDs)

APTES-ZnO QDs were prepared as reported in the literature.23,24 Briefly, 2.8 g of KOH had been dissolved in 50 mL of methanol, flask (A). In addition, 0.55 g Zn (CH3COO)2·2H2O had been dissolved in 25 mL of methanol, flask (B). Both flasks were placed on magnetic stirrers at room temperature (RT) until complete dissolution. The solution of flask (A) was transferred to flask (B) by dropwise addition, until the solution became turbid, indicating the formation of ZnO NPs. The solution was stirred for 1 h. After that, a few drops of 3-triethoxysilylpropan-1-amine (APTES) were added upon stirring until the turbidity of the solution disappeared, indicating the efficient capping of ZnO QDs.

Malvern Zetasizer instrument was used to measure the ζ potential of the synthesized QDs. T80 UV–visible spectrophotometer (PG Instruments, U.K.) was used to record the UV–visible absorption spectra of APTES-ZnO QDs. In addition, LS 55 fluorescence spectrophotometer (PerkinElmer) was employed to assess the photoluminescence (PL) upon excitation at 330 nm. Finally, the high-resolution transmission electron microscope (HRTEM) was beneficial for morphological analysis and measuring particle size. All of the experimental details are described in the SI.

3.3. Synthesis of MgAl-Cl-LDH (Pristine LDH)

Two sets of MgAl-Cl-LDHs have been fabricated by coprecipitation method with subsequent hydrothermal treatment.77 Briefly, MgCl2·6H2O (1.42 g, 7 mmol) and AlCl3·6H2O (0.845 g, 3.5 mmol) were dissolved in 10 mL of Milli-Q water (TDS 4–8 ppm), then added to 30 mL of 0.6 M NaOH solution within 5 s with vigorous stirring to produce LDH1 (L1). Another experiment was performed using (0.61 g, 3 mmol) MgCl2·6H2O, (0.362 g, 1.5 mmol) AlCl3·6H2O in 10 mL of Milli-Q water and 30 mL of 0.3 M NaOH solution to produce LDH2 (F1). After 30 min stirring, to eliminate excess Cl–, the produced LDH slurry was centrifuged two times with Milli-Q water (4500 rpm for 5 min each time). After dispersing the slurry in 30 mL of Milli-Q water, a stainless-steel autoclave with a Teflon lining was used to perform hydrothermal treatment at 100 °C for 6 h, generating a well-dispersed translucent suspension. The whole process was performed under nitrogen atmosphere.

3.4. Synthesis of Pemetrexed Intercalated MgAl-Cl-LDH (LDH-PMX)

Ex situ anion exchange even before hydrothermal treatment is the method employed for PMX loading (intercalation) into LDH.78 The resultant LDH1 (L1) slurry has been resuspended into 30 mL of NaOH solution, 1 M, thus adjusting the pH to 9. PMX (0.03 g, 0.071 mmol) was passed to L1 resultant slurry and stirred for 1 h. After washing twice with Milli-Q water, the suspension was subjected to hydrothermal treatment (100 °C, 6 h) to obtain LDH1-PMX (L2) colloidal suspension with a translucent and homogeneous nature. The same experiment was performed to intercalate the same amount of PMX into LDH2 (F1) to obtain LDH2-PMX (F2). The amount of PMX loaded was determined using a high-performance liquid chromatography (HPLC) assay method and measured at 225 nm, SI.

3.5. Modification of LDH-PMX with Sericin (Seri@LDH2-PMX)

LDH2-PMX (F2) was chosen to be incorporated into sericin protein through the desolvation technique based on X-ray diffraction (XRD), particle size, and ζ potential findings. Briefly, 5 mL of sericin aqueous solution of 1% (w/v) was adjusted to pH 7.4 (using 1 M NaOH) then added to F2 dried powder in 1:1 weight ratio and left on magnetic stirring for 1 h. Then, absolute ethanol (desolvating agent) was dropped (1 mL/min) under constant stirring at RT till the solution became turbid, indicating the generation of sericin NPs. Genipin (GNP) and glutaraldehyde (GTA) were examined based on their effect on the photoluminescence (PL) of APTES-ZnO QDs as bifunctional crosslinkers to achieve NPs stabilization after protein desolvation, thus generating Seri@LDH2-PMX (GNP) (F3) and Seri@LDH2-PMX (GTA) (F4), respectively. The resulting NPs were left overnight under constant stirring till the ethanol evaporated. Two cycles of centrifugation (10 000 rpm, 15 min) were employed for purification. Finally, it underwent redispersion in the original volume of water using a probe sonicator (50% amplitude, 10 min).15,79

3.6. Preparation of APTES-ZnO QDs Conjugated to Sericin-Modified LDH-PMX (APTES-ZnO QDs/Seri@LDH2-PMX)

Different sericin/APTES-ZnO QDs molar ratios were investigated for the luminescence properties of the QDs after chemical conjugation. The chosen sericin/APTES-ZnO QDs molar ratio was 1:3. Accordingly, (0.03 g, 0.16 mmol) EDC·HCl and (0.029 g, 0.16 mmol) K-Oxyma33−36 were added to 5 mL of Seri@LDH2-PMX (GNP) (F3) (equiv to 0.05 g sericin) at RT while stirring for 10 min to make sure that the carboxylic acid groups of sericin were completely activated. Then, 1.7 mL of APTES-ZnO QDs in aqueous dispersion of pH 7.4 (adjusted using 0.1 M HCl) was transferred to that reaction mixture and left for 24 h under stirring at RT. Finally, the NPs were dialyzed against distilled water (dialysis bag with MWCO 12 000–14 000) to remove the unreacted reagents and obtain APTES-ZnO QDs/Seri@LDH2-PMX (GNP) (F5). The same experiment was performed to conjugate APTES-ZnO QDs to Seri@LDH2-PMX (GTA) (F4) to obtain APTES-ZnO QDs/Seri@LDH2-PMX (GTA) (F6).

3.7. Physiochemical Characterization of APTES-ZnO QDs Conjugated to Sericin-Modified LDH-PMX (APTES-ZnO QDs/Seri@LDH2-PMX)

Several methods have been developed to evaluate the physicochemical characteristics of the synthesized nanocarriers. The particle size and ζ potential were measured using Malvern Zetasizer. Transmission electron microscope (TEM) and scanning electron microscope80 were utilized for examining the morphology and surface topography. Moreover, HPLC, FT-IR spectroscopy, XRD, and DSC were very useful for characterizing many features including drug loading. To assess the photoluminescence (PL), an LS 55 fluorescence spectrophotometer was utilized. Stability, redispersibility, and hemolytic activity were also extensively tested. The SI describes all of the specifics.

3.8. In Vitro Drug Release

PMX in vitro release from APTES-ZnO QDs/Seri@LDH2-PMX (GTA) (F6) was compared with that of LDH2-PMX (F2). Apart from the light in a shaking water bath, a certain volume of NPs colloidal suspension (equiv to 2 mg PMX) was placed in phosphate-buffered saline (PBS) pH 7.4 of 30 mL and maintained at 37 °C at 100 rpm. At pH 4.8 PBS, the same procedure was repeated. At predetermined intervals, 2 mL samples of the release medium were centrifuged (10 000 rpm, 10 min). The supernatants were quantitatively analyzed for PMX released while the sediments were dispersed in fresh release medium of 2 mL and returned to the release medium in the shaking water bath to maintain a steady volume. The cumulative amount of drug released over a period of time was plotted against time (h).43

3.9. In Vitro Cytotoxicity Study and Cellular Uptake

As previously reported, MTT assay is a method for assessing the in vitro cytotoxicity. MDA-MB-231 breast cancer cells were used to examine free APTES-ZnO QDs, free PMX, LDH2 (F1), and APTES-ZnO QDs/Seri@LDH2-PMX (GTA) (F6). The details are documented in the SI.81

The in vitro cellular uptake study of APTES-ZnO QDs/Seri@LDH2-PMX (GTA) (F6) and free APTES-ZnO QDs was achieved through analysis of confocal laser microscopy (CLSM) images and quantitative determination by flow cytometry as detailed in the SI.81

3.10. Statistical Analysis

Data analysis is described in the SI.

4. Conclusions

Our study was based on the engineering of magnesium aluminum layered double hydroxides using coprecipitation followed by hydrothermal treatment at 100 °C for 6 h. The PMX drug was then intercalated within the brucite layers of LDH via anion exchange at alkaline pH. After that, sericin protein was utilized to coat the LDH2-PMX through the desolvation technique, followed by chemical crosslinking by glutaraldehyde to stabilize the generated NPs. Finally, the amino groups of APTES-ZnO QDs were chemically coupled to the carboxylic groups of sericin protein. The synthesized nanocarriers are intended to treat and diagnose breast cancer via IV administration. The fabricated NPs were negatively charged (−21.1 ± 0.51 mV) with suitable particle size (201.9 ± 2.30 nm), narrow PDI (0.294 ± 0.02), and high PMX intercalation efficiency (90.70 ± 0.65%). Complete physiochemical characterization, structural identification, and optical, crystallinity, thermal, and morphological analyses were performed for the characterization of LDH, PMX, QD, sericin, LDH-PMX, and APTES-ZnO QDs/Seri@LDH2-PMX (GTA). The spectral analysis was carried out and investigated in detail using FT-IR, UV–visible, photoluminescence (PL), XRD, DSC, ICP, and HRTEM. Moreover, the use of bioconjugation chemical reactions led to the formation of an amide bond between sericin and ZnO QDs. A sustained release manner of the hydrophilic PMX was noticeably exhibited. In vitro cytotoxicity study on MDA-MB-231 breast cancer cells verified our optimized formula’s capability to considerably improve anticancer activity compared to free PMX (IC50 = 1.34 ± 0.03 and 0.31 ± 0.007 μg/mL at 24 and 72 h, respectively). The formula demonstrated in vitro serum stability and acceptable hemocompatibility. Moreover, the prepared NPs were efficiently internalized into MDA-MB-231 cells as evidenced by the cellular uptake study. Also, the study verified that the higher the concentration of guest anion (PMX), the higher its ability to be intercalated within the LDH layers through detailed XRD, particle size, and ζ potential results. However, the proper choice of a crosslinker in a formulation containing an inorganic bioimager is important as it may influence the photoluminescence properties, where GTA crosslinker was more suitable in our study. Finally, it can be concluded that the anticancer efficacy of PMX was markedly improved as it was efficiently taken up by the cancer cells and then retained, compared to the free drug. Sericin enhanced the colloidal stability of LDH that may also justify the efficient cellular uptake. Also, it provides an extra sustained release profile of PMX from F6 which is anticipated to hamper PMX release into circulation while enabling drug release at the tumor site (drug release was about 99.23% after 30 h and 99.3% after 11 h at pH 7.4 and 4.8, respectively). In addition, the integration of APTES-ZnO QDs with the surface of NPs would permit tumor imaging for theranostic applications and could participate in overall anticancer activity via various mechanisms.

Acknowledgments

The authors thank the Science, Technology & Innovation Funding Authority (STDF), Cairo, Egypt, for funding this work through the Innovation Grant (Proposal ID 34857).

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.2c07128.

  • Methodologies, equipment, and software of physicochemical characterizations and in vitro studies (PDF)

Author Contributions

R.M.A.: methodology; investigation; writing—original draft. S.N.K.: supervision; conceptualization; writing—review and editing; resources. S.E.: supervision; validation; formal analysis; resources. K.A.E.: supervision; writing—review and editing; resources. M.T.: validation; formal analysis; methodology. A.A.B.: supervision; resources. M.A.S.: supervision; writing—review and editing. A.O.E.: supervision; conceptualization; resources; writing—review and editing.

The authors thank the Science, Technology & Innovation Funding Authority (STDF), Cairo, Egypt, for funding this work through the Innovation Grant (Proposal ID 34857).

The authors declare no competing financial interest.

Notes

In the future, we are preparing in vivo studies to study the efficacy of the engineered nanocarriers as a promising nanodelivery system for breast cancer theranostics. The analysis of tumor growth biomarkers (such as active caspases and vascular endothelial growth factors (VEGF)), tumor volume, body weight measurement, and tumor histopathology is intended to be done. In addition, the capability for diagnosis would be assessed through in vivo tumor imaging. Also, the organs of higher uptake such as liver and spleen will be analyzed to assess the accumulation of APTES-ZnO QDs to demonstrate their in vivo toxicity.

Supplementary Material

ao2c07128_si_001.pdf (263.3KB, pdf)

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