Characterization and Anticancer Effects of Folate Targeted Inotodiol Liposome From Inonotus Obliquus (Chaga Mushroom)

Inotodiol, one tetracyclic triterpenoid isolated from inonotus obliquus (Chaga mushroom) possess excellent anticancer, antioxidant and anti-inammatory activities, however it has not been applied to medical use due to its low solubility and low bioavailability. Liposome as a good nanodrug carrier with EPR and good biocompatibility has received much research attention. To improve the solubility and bioavailability of IOP, we prepared inotodiol liposomes (IOP-Lps) and folic-acid targeting IOP liposome (FA-IOP-Lps) by ultrasonic method, and their particle size, morphology, zeta potential, entrapment eciency (EE) and drug loading rate (DL) were characterized by DLS, TEM, FT-IR and HPLC respectively. Their in vitro cytotoxicity of human cervical cancer cells HeLa, human liver cancer cells HepG2 and human breast cancer cells MCF-7 were assessed using the MTT assay. The results showed that IOP-Lps and FA-IOP-Lps possess signicant anticancer effects. The results of TEM, FT-IR and DLS conrmed the formation of liposome. The particle size of IOP-Lps were 201.07±6.47 nm and ζ-potential of -50.2±0.5 mV, FA-IOP-Lps 224.33±1.86 nm and ζ-potential of -51.2±0.3 mV, the EE of IOP-Lps and FA-IOP-Lps were 79.14% and 77.33%, respectively. IOP-Lps and FA-IOP-Lps could selectively kill HeLa, HepG2 and MCF-7 cancer cells while nontoxic to normal L02 cells. This is the rst study to give out in vitro information of inotodiol’ s anticancer effects based on nanocarriers. Besides, this drug delivery system is of good sustained release and targeted delivery effects to promote the utilization of both inotodiol and other natural hydrophobic compounds in target treatment of cancers.


Introduction
According to National Institutes of Health (NIH) and National Cancer Institute, cancer is of malignant tumor, a disease which abnormal cell grow with potential to invade or spread other parts of the body.
WHO' s statistic shows that cancer is responsible for an estimated 9.6 million deaths in 2018 and is the second main cause of death in global among which the most common cancers are: breast cancers (2.09 million cases), lung cancers (2.09 million cases), colorectal cancers (1.80 million cases), prostate cancers (1.28 million cases), skin cancer (non-melanoma) (1.04 million cases) and stomach cancers (1.03 million cases), that is approximately 1 in 6 deaths is caused by cancer. About 18 million new cases occured in 2019 and the death number caused by it was about 8.8 million, took up 15.7% of all deaths [1]. Depending on the the patient's health condition and types, locations and grades of cancer, there are different treatment options that includes surgery [2], radiation therapy, chemotherapy, targeted therapy, laser therapy, hormonal therapy, immunotherapy and palliative care [3].
While patients may suffer greater pain in surgery and radiation therapy, they tend to chemotherapy, and it is the primary one among these treatment for over 20 types of cancers. The traditional way of delivering anti-tumor agents is through intravenous administration [4]. This administration leads to low drug concentrations in the diseased area, which leads to poor treatment results and longer treatment cycles.
The low water solubility and bioavailability of these agents often require increased drug doses to achieve the desired therapeutic concentration in the target area, hence, varying degrees of side effects, such as damaging liver cells, causing systemic reactions, and inhibiting the bone marrow hematopoietic system [5], which limits the application of chemotherapeutics to a certain extent.
Targeted therapy, a new form of chemotherapy which targets speci c molecular differences between normal cells and cancer cells. Targeted drugs often work by preventing cancer cells from replicating themselves as they can help prevent cancer cells' dividing and new forming. The rst targeted therapy blocked the estrogen receptor molecule, inhibited breast cancer's growth. Another common example is the class of Bcr-Abl inhibitors, which are used to treat chronic myelogenous leukemia (CML) [6].
To date, targeted therapies are being effectively used for many common cancer types, including liver cancer, breast cancer, lung cancer, bladder cancer, kidney cancer, colorectal cancer, lymphoma, leukemia, pancreatic cancer, skin cancer, prostate cancer, and thyroid cancer as well as other cancer types [7].
Inotodiol, a natural triterpene isolated from Inonotus obliquus, is reported to posses excellent anti in ammatory [8], antioxidant activities and anticancer effects on common cancers including lung cancer, cervical cancer, stomach cancer, liver cancer, breast cancer and prostate cancer [9,10], while doing very little damage to normal healthy liver cells. As inotodiol is a hydrophobic compound, despite that more than ve derivatives [11,12] were synthesized to solve the low solubility, its low bioavailability and the lack of targeting still limit its use. To date, there is no report for inotodiol' s anticancer research based on nanocarriers.
Liposomes, a spherical vesicle of less than 200 nm diameter have at least one lipid bilayer, is a novel drug carrier with good characteristics of biocompatibility, biodegradability and low toxicity, can be used to encapsulate hydrophobic and hydrophilic drug and release the encapsulated drugs continuously [13,14].
Owing to the enhanced permeability and retention effect (EPR), liposome are passive targeting, some ligands can be anchored on the surface of liposomes to realize speci c tumor targeting [15][16][17].
Anticancer drugs that are delivered by tumor targeting vectors form a targeted drug delivery system that can focus the drug at the tumor site and selectively kill tumor cells. This method of administration avoids damage to normal cells and has been extensively studied [18].
Meada and Matsumura rst proposed an enhanced permeability and retention effect (EPR) in 1986. Their study found the phenomenon in which macromolecules (molecular weights greater than 4 kDa) or nanoparticles (10-800 nm) could pass smoothly through the wall space of the tumor vessel due to the good vascular permeability of the tumor tissue [19,16]. Due to the lack of lymphatic re ux of the tumor, the macromolecules or nanoparticles entering the tumor cannot be metabolized, which leads to the accumulation of particles in the tumor. Compared to conventional low-molecular chemotherapeutic agents, large-molecular active substances and nano-active substances are retained in tumors, which enables passive targeting [20,21].
In contrast to passive targeting, active targeting is achieved through the interaction of speci c ligands on drug delivery systems with receptors on their tumor cells [22,23].
Folic acid (pteroyl-L-glutamic acid ) belongs to the vitamin B family and is widespread in green leafy vegetables, it plays an important role as a cofactor in single-carbon metabolizing cells, and is a vitamin necessary for the synthesis of DNA and RNA precursors and cell growth and development [24]. However, folic acid cannot be synthesized by mammalian cells. Reductions in folate carrier and membrane folate receptors both mediate cellular uptake of folic acid. Folate receptor exposure is usually less in adult tissues, but high levels of expression are found in only a few tissues, such as placenta, kidneys, lungs, and choroid complexes. Folic acid receptors are also highly expressed in cancers of epithelial origin, including cervical cancer, ovarian cancer, renal cell carcinoma, breast cancer, colorectal cancer, and choroid plexus [25]. Therefore, the selective overexpression of folate receptors in colon tumors and their high a nity for folic acid provide a unique opportunity for folic acid to be used as a targeting ligand to deliver anticancer drugs to tumor cells. The delivery of these folate-anchored liposome drugs has several advantages over direct binding to chlorophyll. Studies have shown that free folate and folate conjugates can be mediated by folate receptor-mediated endocytosis entry into cells [26,27]. Overexpression of folate receptors is found on the surface of most malignant diseases, but rarely on the surface of normal cells [28,29]. FA has a small molecular weight, simple chemical properties and no advantageous properties of immunogenicity, which makes it suitable for tumor-related therapy. An FA-modi ed carrier loaded with cancer drugs can bind to a folate receptor that is highly expressed on the surface of cancer cells, thereby increasing drug concentration at the tumor site and reducing toxic side effects on normal cells. This makes FA a good ligand candidate in the eld of cancer biotherapy and one of the most popular targeting factors in drug delivery systems.
In order to overcome the problems of low bioavailability and lack of targeting of inotodiol, in this study, we synthesized IOP-Lps and folic acid based targeting liposome FA-IOP-Lps [30], their cytotoxicity to cancer cells and normal were assessed via in vitro experiments, con rming that FA-IOP-Lps has signi cant anti-cancer activity and has minimal damage to normal stem cells. Simulated gastric fluid (SGF) was prepared as Dr. Gao' s method [31]: 23.4 mL 38% concentrated hydrochloric acid and added 100 mL distilled water to prepare diluted hydrochloric acid, 1.64 mL dilute hydrochloric acid and 1 g of pepsin was added to 80 mL distilled water, then diluted to 100 mL with distilled water after they were mixed well.

Isolation of inotodiol
According to Lishuai Ma' s extraction method [32]: 2.0 kg sclerotia of Inonotus obliquus was ground to a ne power, followed by extraction with 60 L 80% ethanol for 2 h at 78 °C for thrice and the ethanol extracts were then mixed, evaporation solvent using rotavapor, the resulting fractions were dried in vacuum, then extracted with petroleum ether (PE), followed by ethyl acetate (EA) extracted. The yield of petroleum ether fraction (PEF) is 38 g.  [34], after sonication, the supernatant were transferred into a 100 mL round bottom ask by pipet, then 15 ml chloroform was added, dealt with ultrasonic for 5 min, evaporate all solvent by rotary evaporation under high vacuum for 12 h, a thick blue lm, that is the IOP liposomes [35]. Finally, 100 mL ultrapure water was added into the ask and sonicated for 5 min to obtain the aqueous dispersion and was kept in 4 °C refrigerator for further use.
Formulations of different materials is list as Table 1. Determination of entrapment e ciency (EE) and drug loading capacity (DL) of IOP-Lps The EE and DL of liposome samples was determined by ultra centrifugation methods [36] and analyzed by HPLC.
The newly prepared liposome was quickly hydrated by 10.00 mL 0.01X PBS then centrifuged at 12,000 g for 30 min. Then the supernatant was analyzed by RP-HPLC (Agilent 1220, USA), the absorbance at 244 nm UV were recorded and peak area were calculated. The amount of free drug was determined by standard calibration curve of peak area. The standard calibration curve was obtained using different concentration of pure inotodiol [37].
Entrapment e ciency (EE) and drug loading capacity (DL) were calculated using the following formula [38]: where M entrapped is the IOP amount entrapped in liposomes, M total is the total IOP amount in the resulted preparation and M Lps is the amount of total IOP in the resulted preparation.
In vitro release of of IOP -Lps Page 7/16 2 mL of IOP-Lps was suspended in a MW 3500 dialysis bag [39,40] (activated by boiling water for 15 minutes) and then immersed into beaker with either 50 mL simulated gastric uid (SGF), pH 5.5 phosphorous buffer and pH 7.4 PBS, each with 0.5% (v/v) Tween-80, and stirring at 37 °C under 200 rpm [47], beakers were wrapped with tin foil to protect them from light.

Characterization of of blank liposome, IOP-Lps and FA-IOP-Lps
IR spectra of blank liposome, IOP-Lps and FA-IOP-Lps The infrared (IR) spectra of inotodiol, blank liposome, IOP-Lps and FA-IOP-Lps were characterized by potassium bromide (KBr) tableting method [41] using Bruker infra-red spectrophotometer (TENSOR 27, Germany). A tiny drop of hydrated IOP-Lps and was drop onto the surface of potassium bromide tablet, the transmittance within the wave number range of 4000-400cm -1 was measure after drying by infrared drying oven.

Morphology of IOP-Lps
One drop of IOP-Lps was taken and dropped on the copper net by the method of phosphotungstic acid negative staining [42]. After natural drying, it was negative stained with 1% phosphotungstic acid for 2min. After drying, the morphology of samples was characterized with transmission electron microscopy (TEM, JEM-2100F, JEOL, Japan).
Particle size and zeta potential of IOP-Lps and FA-IOP-Lps The average particle size and zeta potential of samples was detected by dynamic light scattering (DLS) method by Malvern Zetasizer (Nano ZS 90, Malvern Instruments, UK) at room temperature [35]. Samples were suspensions with concentration below 1 mg/ml.
All procedures were carried out in triplicate.

Culture of HepG2, L02, MCF-7 and HeLa cells
The frozen HepG2, L02, MCF-7 and HeLa cells were taken out from the liquid nitrogen tank and resuscitated rapidly, they were cultured with the medium consist of 90% DMEM, 10% (v/v) FBS plus an extra 1% (v/v) penicillin/streptomycin under standard incubation condition (37 °C, 5% CO 2 ). The cells were carried out to subculture for 3 times until the growth condition is stable.

In Vitro Cytotoxicity of inotodiol, IOP-Lps and FA-IOP-Lps
The cytotoxicity of cancer and normal cells were assessed using MTT assay. HepG2, L02, MCF-7 and HeLa cells (1 × 10 4 cells per well) were seeded in a 96-well plate and incubated overnight under standard incubation condition (37 °C, 5% CO 2 ). Various concentrations of samples were added into each well to replace the original medium, followed by incubating for 12 h, 24h, 36h under standard incubation condition. Afterward, MTT solution (5 mg/mL, 20 μL) was added into each well and further incubated for 4 h. Finally, after the removal of old media and the generated formazan were dissolved with DMSO under mild shaking, the OD value at 490 nm (570 nm as reference) of each well were immediately measured by an enzyme-linked immunosorbent assay reader [43] to calculate the viability and IC 50 .

Statistical analysis
In this experiment, datum were tested three times unless otherwise stated. ANOVA (analysis of variance) were used for statistically signi cance. IC 50 value were obtain from SPSS software. Data are expressed as mean ± standard deviation (SD).

Results And Discussion
Encapsulation e ciency (EE) and drug-loading capacity (DL) of IOP-Lps It could be seen from Table 2 that under the selected factors and level conditions, the optimal combination condition was a3d2b1c3, and the in uence degree of each factor on the encapsulation rate was A D B C, i.e. 5.00 mg cholesterol, 10.65 mg DPPC, 1.20 mg DSPE-PEG 2000 , 1.00 mg IOP. The highest encapsulation e ciency of liposomes was 77.33%.
As shown in Table 2, the EE of IOP-Lps with different content was obtained. Overall, the EE of the nanocarriers mostly affected by cholesterol content. The EE gradually grows large as cholesterol content increased from 2.50 mg to 5.00 mg, However, as cholesterol took up too much bilayer space, it will lead to membranes' rigidity gradually increased with uidity decreased, besides, adding cholesterol may reduce the ratio of phospholipids used to form liposomes at the same time, which might lead to the leaking of content drug, this indicating that only certain amount of cholesterol could enhance the phospholipids in bilayer [35].
The EE and DL of IOP were 77.33% and 35.55%, respectively. Particle size, zeta potential and morphology of IOP-Lps and FA-IOP-Lps The particle size of the sixth group with the highest encapsulation e ciency was measured. As shown in Fig. 3a and 3b, the Z-average of IOP-Lps was 254.7 nm and the zeta potential was -50.2 mV before passing through the membrane. After sonication and ltered with 0.22 μm needle lter, the Z-average decreased to 201.07±6.47 nm and the zeta potential was -59.8±7.3 mV. The Z-average of FA-IOP-Lps was 224.33±1.86 nm and zeta potential was -54.6±5.7 mV (measured after sonication and ltered with 0.22μm needle lter). The hydrated liposomes were easy to agglomerate, to a certain extent the ultrasonic treatment inhibited the aggregation, the decrease in Z-average also attributed to the elimination of particles whose size exceeds 220 nm, so that the average value is reduced. According to Hadian, the liposome of <−30 mV zeta potential are considered as optimum formulation, showing that the obtained liposome is relatively stable. Besides, the polydispersity index (PDI) was 0.388±0.017 in 0.01x PBS indicate relative low variance, and liposomes were well dispersed [44,45].
Transmission electron microscopy (TEM) was used to showed its morphology. It can be seen from the Fig. 3c, 3d, 3e that the IOP-Lps are irregular or nearly spherical, well dispersed, but slightly agglomerated. These results con rmed that IOP were successfully coated into phospholipid.
It is worth noting that the liposome particle size seen under TEM was about 100nm, which was signi cantly smaller than the data measured by DLS, this experiment was done in triplicate and of different concentration to avoid the in uence of concentration, this phenomena might be attributed to PEGylation [33,46], PEG was not successfully dyed by phosphotungstic acid, therefore it could not be observed under TEM.
Infrared characterization As Fig. 3f has shown, the characteristic absorption peak of blank liposome, 2930 cm -1 was the saturated C-H stretching vibration absorption peak, 2850 cm -1 was the CH 2 symmetric stretching absorption peak, 1740 cm -1 was the stretching vibration of C=O in phospholipid, the contrast showed that due to the in uence of 1630 cm -1 , the absorption of C=O in 1740 cm -1 became stronger, and the absorption frequency of C=O in the two substances increases with the overlap of C=O absorption peak, and there was an absorption peak at 1360 cm -1 , but there was no such absorption peak in the blank liposome. It was speculated to be the result of the angular vibration (1410 cm -1 ) of -CH 2 -in IOP moving to lower wavenumbers. Therefore, IOP was successfully incorporated into the liposome [47,48].

In vitro release
The dialysis method was used in the experiment to detect the release of IOP in SGF, pH 5.5 PBS and 0.01x pH 7.4 PBS. As shown in Fig. 3g, the free IOP release rates in SGF, pH 5.5 PBS and pH 7.4 PBS were 89.51 ± 0.56%, 84.59 ± 0.53% and 83.18 ± 0.47% respectively, and IOP-Lps release rates in SGF, pH 5.5 PBS and pH 7.4 PBS were 57.02 ± 0.43%, 49.57 ± 0.63% and 50.98 ± 0.62% respectively. The release speed was relatively large at the initial 24 h, and subsequently slowed down, as reported in previous studies. A large burst in the initial phase was mainly due to the separation of the drug from the outer surface of the lipid; the subsequent slow release was mainly due to the diffusion of the drug across the liposome bilayer into the release medium [49]. Therefore, liposomes can effectively slow down the release of drug.

Cellular cytotoxicity assay
To evaluate the cytotoxicity of different concentrations of IOP-Lps to cancer and normal cells, MTT assays were carried out to determine cell viabilities.
Hela, HepG2, MCF-7 and normal Hepatic L02 cells were treated with full culture medium as negative control, IOP as positive control, IOP-Lps as control group of different concentration.
The HepG2 cell viability as list in Fig. 4. From Fig. 4 the cell viability revealed that the FA-IOP-Lps had the best inhibition rate of HepG2 cells. And the living cells under microscope in Fig. 5 also con rmed that liposome signi cantly promote IOP' s inhibition of HepG2 cells.
The IC 50 values of IOP, IOP-Lps and FA-IOP-Lps are shown in Table 3a,b,c. Liposomes have the same phospholipid bilayer structure as cell membranes [50]. This structural feature promotes the ability of cells uptake of IOP in liposomes. At the same time, folate receptor (FR) overexpression [29] in cancer cells, it works along with enhanced permeability and retention effect (EPR) to promotes FA-IOP-Lps to recognize and accurately target cancer cells.

Conclusions
In this study, the inotodiol liposomes IOP-Lps and FA-IOP-Lps possessing signi cant anticancer properties were prepared, the synthesized optimum FA-IOP-Lps were of considerable particle size (Z average 224.33 nm), good stability (zeta potential -54.6 mV), uniform spherical morphology, good entrapment e ciency (77.25%) and mono disperse condition (PDI) which are highly recommended for the effective in vitro and in vivo drug release. With the 48h IC 50 value of 82.09 and 74.56 µmol/mL for HepG2 (liver hepatocellular cells), respectively, con rming that coating FA modi ed PEG, liposomes could control the release of IOP and enabled active target delivery to treat cancer cells and enhance intracellular uptake, while it shows very low toxicity to L02 normal healthy cells. It is a controllable programed of targeted chemotherapy. The in vitro experiment results exhibited its excellent anticancer e ciency and be a powerful targeted chemotherapy for destruction of cancers. This is the rst in vitro study for inotodiol liposome and provide scienti c information for further study for the application of intodiol. The signi cance of this study is that these ndings demonstrated FA-IOP-Lps signi cantly increased bioavailability of inotodiol, we hope this study will contribute to further research in vivo, and help those patients who are suffering from cancers.

Declarations
Ethics approval and consent to participate Not applicable

Consent for publication Not applicable
Availability of data and materials Both the materials and the datas generated and analyzed during the present study are available from the corresponding author on reasonable request.
Competing interests There are no con icts to declare. Authors' contributions Yangpeng Lu contributed to the design of experiments, methodology, veri cation, software, formal analysis, in vitro release studies, data analysis and write manuscript. Xudong Gao and Zhongqin Chen contributed to the cell culture and cytotoxicity assay, Zihan Xue, Yanan Jia Nannan Li and Yajie Wang contributed substantially to data acquisition, statistical analysis, and data interpretation.
Haxia Chen has made an vital contribution to the supervision of the manuscript and funding the experiment. All authors read and approved the nal manuscript.