Plant Extracts-Based Nanocarriers for anticancer therapy: Comparison
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Please note this is a comparison between Version 2 by Jason Zhu and Version 1 by Abdulrahman Elbagory.

Nanocarriers enhance the dissolution and bioavailability of drugs and facilitate their targeting effect. Taking the potential toxicity into consideration, the incorporation of natural “green” materials, derived from plants, in the nanocarriers fabrication, improve their safety and biocompatibility. These green components can be used as mechanical platforms, targeting ligands or can play be involved in the synthesis of nanoparticles. 

  • green nanotechnology
  • nanocarriers
  • plant extracts

1. Introduction

Nanotechnology is an interdisciplinary field of science concerned with the processing of matter at atomic/molecular level [1]. Nanotechnology is applicable in many areas such as the chemical industry, pharmaceutical industry, optics, electronics, energy science and biomedical sciences [2]. The size of the nanoparticles (NPs) is comparable to the size of proteins and various intracellular macromolecules, which allows them to take advantage of cellular machinery to assist in the drug delivery [3]. Additionally, in comparison to bulky materials, NPs have large surface to volume ratio allowing for chemical modifications to tune their properties [4]. NPs can be used as nanocarriers to encapsulate drugs or biomolecules inside their structures and/or absorb them on their surfaces. There are different types of nanocarriers including polymeric NPs, liposomes, micelles, dendrimer, hydrogel, mesoporous, 0, 1 and 2-D materials [5]. These types are broadly divided into organic, inorganic and hybrid nanocarriers [6]. Several types of payloads can be delivered using NPs such as conventional drugs, polypeptides, proteins, vaccines, nucleic acids, genes, etc. [7].

The commonly used conventional chemotherapies include alkylating agents, antitumour antibiotics (e.g., epirubicin, doxorubicin (DOX)), antimetabolites (e.g., 5-fluorouracil (5-FU), methotrexate, gemcitabine), topoisomerase inhibitors, and mitotic inhibitors (e.g., paclitaxel and docetaxel) [8]. Chemotherapy drug delivery for local and metastatic tumours is associated with various drawbacks. Such problems may include high toxicity in normal cells, lack of tumour target selectivity, high volume drug distribution and rapid drug clearance [9]. Nanocarriers can improve the safety and efficiency of drugs by increasing their water solubility and stability, enhance their circulation time, improve their uptake by targeted cancer cells or prevent their enzyme degradation [10]. The current reports on the use of nanocarriers for drug delivery focus on: (1) the choice of suitable carrier materials to achieve high drug encapsulation rate and controlled and targeted release speed; (2) improvement of targeting ability via surface functionalization; (3) augmentation of drug biological activity with using carrier materials of similar activity; (4) formulating responsive nanocarriers that are able to release the loaded drugs at designated sites in a response to the local environment (e.g., pH-response release and response to enzymatic degradation of nanocarriers, etc.); (5) performing in vitro and in vivo assays to compare the biological activity between the loaded drugs and their free forms and to assess the safety of the nanocarriers and their stability. The use of nanocarriers inside the body will allow them to interact with blood components and vessels, normal tissues, etc., meaning that they can influence human health and therefore it is important to consider the safety of the components included in the synthesis of these nanomaterials. Despite advancements in nanocarriers, their transformation in medical applications remains insufficient. This mainly is due to their lack of biodegradation, instability in circulation, poor bioavailability, long-term potential toxicity, and inadequate tissue distribution.

Therefore, the overall aim of including green components in the fabrication of NPs as nanocarriers is to decrease toxicity to the body, to have an environmentally benign industry process and to increase affordability. The green synthesis may be facilitated by plants extracts and microbes or their isolated biomolecules [11]. The green based nanocarriers are mainly synthesized by the bottom-up approach within three fundamental conditions of synthesis. These conditions are based on the selection of a green non-toxic solvent, coupled by a good reducing agent, and thirdly incorporating an efficient stabilization material.

2. Plant Extracts-Based Nanocarriers for anticancer therapy 

The use of plant extracts in the biosynthesis of different types of NPs has been widely explored due to their availability, renewability, easy and safe handling compared to other green routes such as bacteria or fungi. The presence of effective phytochemicals with different functional groups such as ketones, aldehydes, flavones, amides, terpenoids, carboxylic acids, phenols, and saponins can reduce metal salts into MNPs without the need for chemical stabilizers/capping agents.
Biodegradable poly(D,L-lactide) NPs were synthesized using five plant extracts namely, Syzygium cumini, Bauhinia variegata, Cedrus deodara, Lonicera japonica and Eleaocarpus sphaericus as stabilizers and emulsifiers. The synthesis was done by sonicating poly(D,L-lactide) solution with dichloromethane and the aqueous plant extract. The transmission electron microscopy (TEM) analysis showed that the produced poly(D,L-lactide) NPs are spherical in shape and with different average sizes depending on each plant extract used. The poly(D,L-lactide) NPs from Lonicera japonica were uniform and therefore were selected for the loading of quercetin as a model for delivery of anticancer drug. The loading of quercetin was confirmed by the reduction of its absorption at 350 nm. The in vitro release study using phosphate buffer showed a burst release of around 20–27% of quercetin in 30 min, followed by controlled release of 32% after 24 h [12]. Similar study reported the green synthesis of PLGA NPs in a solvent-free method using castor oil derivative (acrysol oil). The study also incorporated resveratrol into the green PLGA-NPs which showed enhanced cytotoxicity against MCF-7 cells [13][12].
Mukherjee et al. (2012) used the aqueous extract of Eclipta alba to formulate spherical GNPs. The synthesis was optimized using different volumes of the plant extract. The GNPs showed efficient in vitro stability when incubated with different buffer and biological solutions. The biosafety of the GNPs (up to 114 µM) was shown by maintaining the viability of MCF-7 and MDA-MB-231 after 48 h incubation. However, 10 µM of the GNPs loaded with DOX exhibited 50% cell viability reduction of MCF-7 cells after the same period [14][13].
Biocompatible GNPs and silver nanoparticles (AgNPs) were synthesized using the leaf aqueous extract of Butea monosperma. The reaction was optimized by varying the leaf extract volume and keeping the volume of the gold and silver salts unchanged, which produced NPs after 5 min and 2 h for GNPs and AgNPs, respectively (Figure 1). The study reported the use of AgNO3 staining to confirm that low molecular weight proteins content of B. monosperma were responsible for the synthesis and stabilization of the produced MNPs. Dynamic light scattering (DLS) analysis confirmed the conjugation of DOX into the biosynthesized GNPs and AgNPs with the increase in their hydrodynamic size. Both MNPs loaded with DOX exhibited higher cytotoxicity compared to the pristine drug against mouse melanoma (B16F10) cells. Further, the fluorescence microscopy indicated higher red fluorescence in case of the DOX loaded into the MNPs compared to the free DOX, this proves higher internalization of DOX inside the B16F10 cells when loaded into the MNPs [15][14].
Cancers 13 05686 g004 550
Figure 1. The characterization of the MNPs showing the X-ray diffraction peaks (a,b) confirming the crystallinity of the GNPs (denoted b-Au-500) and AgNPs (denoted b-Ag-750) from B. monosperma. c-f are the TEM images of the GNPs and AgNPs synthesized using different volumes of the B. monosperma extract 250 µL (c), 500 µL (d) for GNPs and 500 µL (e), 750 µL (f) for AgNPs. Reproduced from [15][14].
DOX was also loaded into GNPs biosynthesized using the aqueous extract of Peltophorum pterocarpum. The study explored the cytotoxicity of the loaded DOX in mice injected with B16F10 cells. The results displayed a substantial time-dependent reduction of the tumour volume in the mice treated with the loaded DOX-GNPs in comparison the free DOX treated mice [16][15].
The eggplant fruit extract was also reported in the reduction and stabilization of GNPs using the irradiation of natural sunlight. The produced GNPs were functionalized wit HA as a targeting agent. The carboxylic groups of HA were activated by 1-ethyl-3-(3-dimethylaminopropyl) carbodi-imide and N-hydroxysulfosuccinimide to allow its conjugation with the amid groups of the anticancer drug, metformin. The release of the loaded metformin was found to be negligible at higher pH, citing the targeting effect of the nanocarrier. Moreover, the loaded metformin showed higher cytotoxicity compared to the free drug. The MTT results after 48 h gave IC50 value of 4 µg/mL against HepG2 cells for the loaded metformin compared to 10 µg/mL for the free drug. The authors used the acridine orange and ethidium bromide staining experiment and showed that loaded metformin induce cell death via apoptosis. Interestingly, the same experiment showed no change on the viability of mouse embryonic fibroblast (NIH 3T3) cells, which was attributed to the targeting effect of HA to the cluster determinant receptor (CD-44) that is expressed on HepG2 but not on NIH 3T3 cell line [17][16].
Ganeshkumar et al. (2013) reported the use of fruit peel extract of Punica granutum (pomegranate) in the synthesis of stable GNPs. The authors coupled FA as targeting ligand and the coupling was confirmed by a red shift in the UV-Vis spectrum of the GNPs. After, 5-FU was loaded into the GNPs, which was shown by the quenching of the 5-FU fluorescence. It was also found that the nanocarrier was safe up to concentration of 750 µg/mL by evaluating the morphology, hatching and survival rate of the Zebrafish embryos. To utilize the targeting effect of FA against breast cancer, the cytotoxicity of nanocarrier loaded with the 5-FU was done against MCF-7 cell line and the MTT results gave IC50 value (250 ng/mL) three times lower than the IC50 value of the free 5-FU (1000 ng/mL). The Western blot analysis has confirmed that nanocarrier loaded with 5-FU induced apoptosis via G0/G1 cell cycle arrest [18][17]. The 5-FU loaded on GNPs formulated from the Borassus flabellifer fruit extract also showed higher cytotoxicity against the pancreas cancer MiaPaCa-2 cell line than free 5-FU as exhibited by the MTT assay [19][18].
Almond seed water extract was also used to biosynthesize GNPs. The biosynthesized GNPs were capped using polyethylene glycol 9000 (PG9) before the functionalization with quercetin as an anticancer drug model. The MTT results against MCF-7 showed that only the biosynthesized GNPs functionalized with PG9 and quercetin induced cell death, whereas quercetin alone and the green GNPs without PG9 or quercetin did not show significant antiproliferative activity [20][19].
The biosynthesis of AgNPs was also reported using the fruit water extract of the Indonesian Garcinia mangostana. The optimization was done by changing the concentration of AgNO3, temperature and the pH conditions. The loading of protocatechuic acid on the AgNPs resulted in significant antiproliferation activity (80%) against HCT116 colorectal cells compared to (5%) from the free AgNPs at concentration of 15.6 µg/mL. The IC50 of the free protocatechuic acid was 15 times higher than the IC50 of the loaded protocatechuic acid against the same cell line. The authors found that the cytotoxicity of the AgNPs loaded with protocatechuic acid was attributed to the loss of mitochondria’s membrane potential and the generation of ROS [21][20].
Cai et al. (2020) reported the synthesis of magnetic nanocomposites of Fe3O4 NPs facilitated by Euphorbia cochinchensis leaf extract. The resulted NPs were then incorporated with mesoporous silica and modified with carboxyl groups to prevent their agglomeration. The green nanocomposites were then loaded with DOX and showed high release at acidic pH. With the aid of external magnetic field, the authors concluded that these nanocomposites could be utilized for targeted and controlled release of DOX at tumour site [22][21].
The synthesis of ZnONPs was also facilitated by the tea ethanolic extract. The NPs were then loaded with chitosan to facilitate the targeting effect and were then loaded with paclitaxel. The nanocarrier showed selective toxicity against MCF-7 cells with minimum effect on normal fibroblasts compared to the free paclitaxel [23][22].

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