Novel Multifunctional Magnetic Nanoparticles：An Efficient Theranostic Platform for Magnetic Resonance Imaging and Targeted Therapy of Cervical Cancer


 Background: The high incidence and mortality rates of cervical cancer pose a serious threat to women's health. Traditional chemotherapy has inevitable drawbacks of nonspecific tumor targeting, high toxicity, and poor therapeutic efficiency. In order to overcome these shortcomings, a novel multifunctional magnetic nanoparticles drug delivery system with tumor targeting and magnetic resonance imaging was developed to achieve precise diagnosis and targeted tumor killing effects.Methods: Transmission electron microscopy, dynamic light scatting and ultraviolet methods were used to characterize the nanoparticles in vitro. Cell function tests were performed by scratch, transwell and flow cytometry assays. MTT was used to detect the toxicity of the nanoparticles. The motion trajectory, drug release and uptake studies were carried out in vitro. The in vivo pharmacokinetic and drug distribution studies were verified by high performance liquid chromatography methods. Attenuation of the MRI signal by the nanoparticles and their enhanced antitumor efficiency were examined in vivo in mouse cervical cancer models. Sequencing and proteomics were used to detect the key antitumor molecules of the nanoparticles.Results: Multifunctional magnetic nanoparticles coated with ferric oxide nanoparticles and doxorubicin hydrochloride (DOX-Fe3O4-PEG-PLA-NPs) was prepared successfully. No toxicity was detected of PEG-PLA-NP, however, the tumor killing effect was enhanced under the alternating magnetic field significantly. The drug-release study showed that the cumulative release rates of NP groups were much less than free DOX group, while the drug release rate increased under acidic condition. In addition, DOX-Fe3O4-PEG-PLA-NPs showed improved internalized into carcinoma cells under magnetic field significantly. In vivo studies demonstrated that the combined therapy under an alternating magnetic field displayed improved therapeutic effect when compared with individual therapies as documented by the delayed tumor growth, inhibition of metastasis, and prolonged survival. The in vitro and in vivo MRI results showed that the multifunctional magnetic nanomaterial had a better MRI signal reduction effect and a higher T2 relaxation rate.Conclusions: We developed an cervical cancer targeting nano-carrier drug delivery system successfully, which showed perfect excellent T2 contrast magnetic resonance imaging, chemotherapy-sensitizing, tumor targeting , and anti-tumor effect, thus have the potential to be a new theranostic strategy for ovarian cancer patients.


Background
Cervical cancer is the fourth most common cancer in women, with approximately 570 000 new cases and 311 000 deaths occurred in 2018; however, the incidence of cervical cancer is particularly high in China, with 106 000 new cases and 48 000 deaths [1,2] . Currently, cervical cancer treatment consists of surgery or chemoradiotherapy for early-stage disease and concurrent chemoradiation for advanced-stage patients. However, these conventional treatment strategies are highly aggressive or nonspeci c and often accompanied by serious side effects [2] . The above traditional treatment regimen has not signi cantly improved the prognosis of patients or prolonged patient survival [3] . Therefore, new treatment strategies are urgently needed for the diagnosis and treatment of cervical cancer. Doxorubicin (DOX) is an anthracycline glycoside antibiotic originally produced by Streptomyces peucetius var. caesius [4] . DOX exerts its cytotoxic effects as a DNA intercalating agent to inhibit DNA and RNA biosynthesis. Thus, DOX is widely used as either a single agent or in combination with other chemotherapeutic regimens to treat various kinds of solid tumors as while as shown substantial treatment potential and is regarded as one of the most potent FDA-approved chemotherapeutic drugs [5] . However, its application has been limited by its poor water solubility, high toxicity, and side effects such as cardiotoxicity, hepatotoxicity and the suppression of bone marrow hematopoietic function [6] . The high rate of DOX toxicity has prompted the development of alternative treatment strategies to reduce the drug's serious side effects. This includes the use of encapsulated drug delivery by various nanocarriers, the new drug delivery systems and administration routes for DOX to increase its tissue selectivity and reduce its toxicity pro le.
Early accurate diagnosis and precise targeted tumor therapy strategies are the key factors to improve the prognosis of patients and signi cantly prolong the survival time of tumor patients. A large number of articles have reported on the use of nanomaterials to carry chemotherapy drugs for the treatment of cervical cancer and showed excellent tumor killing effect, such as the codelivery of chemotherapeutic agents by nanocarriers [7] , gene therapy [8] , and recombinant protein therapy. In recent years, advances in nanoscience and nanomedicine have led to the development of multifunctional integrated nanocarriers for molecular and cellular imaging, speci c tumor therapy, and cancer detection and screening [9][10][11] .
Magnetic nanoparticles (MNPs) have been investigated for decades as drug delivery systems because of their high magnetic responsiveness, biodegradability, biocompatibility [12] , high delivery e ciency and potential targeting functions. Iron oxide nanoparticles (IONPS) are considered candidates for biomedical applications since they ( ) possess unique magnetic properties derived from their ability to strengthen tissue protons to relax, resulting in enhanced Magnetic resonance imaging (MRI), ( ) With nanomotor characteristics under external magnetic eld attraction, it can autonomously move to the target site and increase its tumor tissue penetration ability; ( ) Generating magnetic hyperthermia(MH) and reactive oxygen species (ROS) under the alternating magnetic eld; and ( ) are easily modi ed to adjust their physicochemical parameters to function at the cellular level. Moreover, iron oxide nanoparticles are the only MNPs approved for clinical use by the US Food and Drug Administration (FDA) [13,14] . Iron oxide nanoparticles (IONPS) are considered to be a suitable candidate for biomedical applications because of their molecular transport [15] , anemia [16] , magnetic targeted therapy [17] , MRI [18] and hyperthermia [19] properties.
In this study, we successfully constructed a novel PEG-PLA-based polymeric nanocarrier co-encapsulated with DOX and IONPS using a single emulsion evaporation method. The motion and magnetic targeting ability of the material was investigated through its motion track in vitro. The slow-release properties of multifunctional magnetic nanoparticles were analyzed by in vitro release experiments, pharmacokinetic experiments and tissue distribution studies. The in vitro uptake and tissue distribution experiments con rmed the magnetic targeting of this multifunctional nanoparticle to tumor cells and tissues. Cell function experiments, toxicity experiments, anti-tumor e cacy studies, and sequencing results showed that the tumor-killing mechanism of multifunctional nanoparticles were that magnetic nanoparticles generate magnetic heat and active oxygen with tumor-killing ability under the action of an alternating magnetic eld. The synergistic effects of magnetic heat, reactive oxygen species and chemosensitization promote the apoptosis of tumor cells, and then realize the killing effect of tumor cells. In addition to verifying the sustained release effect, tumor targeting properties and excellent anti-tumor effects of multifunctional magnetic nanoparticles, we also investigated the ability of multifunctional magnetic nanoparticles as contrast agents in MRI imaging. Taken together, this work not only presents evidence showing that DOX-Fe 3 O 4 -PEG-PLA-NPs is a potentially viable therapeutic paradigm to cancer therapy; It also suggests that IONPS promise in offering emerging therapies to revolutionize nanomedicine in the diagnosis and treatment of a myriad of human diseases.

Synthesis and Characterization of DOX-Fe 3 O 4 -PEG-PLA-NPs
As outlined in Figure 1a, the ow chart of DOX-Fe 3 O 4 -PEG-PLA-NP, which indicates the successful preparation of PEG-PLA multifunctional nanocarriers containing magnetic Fe 3 O 4 and DOX at the core using oil-in-water emulsion and subsequent solvent evaporation methods including two steps: 1) synthesis of amphiphilic polymeric nanocarrier PEG-PLA, and 2) DOX and IONPS are encapsulated into PEG-PLA nanomaterials. As visualize by transmission electron microscopy (TEM), PEG-PLA-NP, DOX-PEG-PLA-NP and Fe 3 O 4 -DOX-PEG-PLA-NP exhibited uniform particle size and excellent dispersion. As measured by dynamic light scattering (DLS), the size of PEG-PLA-NP is 50nm, while DOX and Fe 3 O 4loaded increase the size of DOX-PEG-PLA-NP and Fe 3 O 4 -DOX-PEG-PLA to 90 nm and 120 nm respectively, the particle sizes of the three nanocarriers were similar to those measured by transmission electron microscopy ( Figure 1b). As showed the uorescence spectra of FITC coupled with different nanocarriers. The excitation wavelength is 488 nm, and the emission wavelength ranges from 500 nm and 750 nm ( Figure 1c). Figure 1d shows the UV absorption spectra of PEG-PLA-FITC empty nanoparticles (blue), PEG-PLA-FITC containing DOX nanoparticles (red), and PEG-PLA-FITC containing Fe 3 O 4 and DOX nanoparticles (black). Figure 1e shows the infrared spectra of the PEG-PLA-FITC, PEG-PLA-DOX-FITC and Fe 3 O 4 -DOX-PEG-PLA-FITC nanoparticles. MTT assay was used to detect the cytotoxicity of PEG-PLA-NP to cervical cancer cells (Hela) at different concentrations and for different duration. The results showed that the cytotoxicity gradually increased with the increase of the concentration. When the concentration was 100µg/ mL and the action time was 72 h, the survival rate of the tumor was 59.02%. MTT results con rmed the low toxicity of PEG-PLA nanomaterials (Figure 1f). Figure 1g shows the magneto-thermal transformation of different Fe 3 O 4 concentrations(100-1000 µg/mL) under the alternating magnetic eld, which shows concentration-dependent and time-dependent. When the concentration of Fe 3 O 4 is 1000 µg/mL, the temperature is 56.3±0.374℃, which is much higher than that of H 2 O (30.367±0.379℃).

Encapsulation rate and drug loading
The encapsulation rate is de ned as the weight percentage of DOX in the PEG-PLA-NPs. The DOX encapsulation and drug loading rates into the DOX-Fe 3 O 4 -PEG-PLA-FITC micellar nanoparticles were 79.9% and 23.4%, respectively. The encapsulation and drug loading rates into the DOX-PEG-PLA-FITC nanoparticles were 75.5% and 21.9%, respectively.
Drug release pro le in vitro pattern. In general, after the same time and at the same pH, the release e ciency of each of the nanocarrier groups was much lower than that of free DOX group, but there was no signi cant difference between the two drug-loaded nanomicrogroups, which proved the sustained release effect of the nanocarriers in vitro. The drug release rate of DOX-NP S at pH 7.4 was slower than that at pH 6.5, demonstrating the pH sensitivity of the nanocarrier. After incubation for 160 h, approximately 35% of the drugs were released at pH 6.5, while the release rate was 25% at pH 7.4.

Pharmacokinetics
The standard curves of DOX in tissue and plasma samples were established, and linear regression analysis obtained a linear regression equation between the DOX concentration (X) and the area under the curve (Y) ( Figure S1). The pharmacokinetic characteristics are shown in Figure 2c The elimination periods (T 1/2 ) of DOX-Fe 3 O 4 -PEG-PLA and DOX-PEG-PLA were 15.92 h and 13.47 h, respectively, which were signi cantly longer than that of the free DOX group (6.41 h, p <0.05). The AUCs of DOX-Fe 3 O 4 -PEG-PLA and DOX-PEG-PLA were 159.81 (µg/mL/h) and 132.72 (µg/mL/h), respectively (p >0.05), which were approximately 3 times that of free DOX group (µg/mL/h) (p <0.05). The elimination period (T 1/2 ) in the nanocarrier groups were signi cantly greater than that of the free drug group, and the AUC values were much greater than that of the free DOX group. Based on these results, given the same amount of drug, the drugs in the nanocarrier groups can circulate in the body and act for a longer time, thus having a stronger killing effect on tumors. In addition to the results of the drug release studies, the pharmacokinetics also con rmed the slow release effects of the nanocarriers. under AMF the cell inhibition rates of the three groups were 77.38%, 80.18%, 88.04%, respectively. The above results all prove that the alternating magnetic eld improves the tumor cell killing effects of the magnetic nanocarrier. Interestingly, the killing ability of the free DOX group increased rapidly after 8 h of treatment, while the killing ability of the two nanocarriers increased rapidly after 48 h of incubation. This further proved the sustained release effect of the drug-loaded nanocarriers. Figure 3b shows the ability of different groups to produce reactive oxygen species (ROS) with or without alternating magnetic eld. Blue uorescence represents DAPI staining nucleus, and green uorescence represents ROS content.

Cellular cytotoxicity of the nanocarriers in vitro
In vitro functional test

Cellular uptake results in vitro
The results of the cellular uptake experiment are shown in Figure 5. DAPI was used to stain the nucleus to detect the cell density of each group. The results showed that there was no statistically signi cant difference between the groups (p > 0.05), indicating the same number of cells in each group. and 6f), the tissue content of DOX in the free DOX group was higher than that of the nanocarrier group at all time points. Free DOX is more likely to accumulate in the reticuloendothelial system, increasing spleen and kidney toxicity. Interestingly, at different time points, the content of DOX in the blood of the free DOX group was lower than that of the nanocarrier group. The nanocarrier group had more drug circulating in the blood and a more durable tumor-killing effect. Figure 6j shows the Prussia blue staining in tumor from mice with the DOX-Fe 3 O 4 -PEG-PLA-NP group treatments.

Antitumor effects of the different groups in vivo
The schematic diagram of treatment plans in subcutaneous tumor-bearing mouse models with different treatments (Figure 7a). Image taken with IR thermal camera of Hela tumor-bearing mice with different treatments under AMF (Figure 7b), and Figure 7c shows the tumor temperature curves of tumor region under AMF. Figure 7d shows images of the tumor-bearing mice after 26 days of treatment, as well as the tumor map of the mice. The results show that the volume signal from tumor-bearing mice in the DOX-Fe 3 O 4 -PEG-PLA-NP group treated with the alternating magnetic eld is relatively weak, which proves that DOX-Fe 3 O 4 -PEG-PLA-NP has a strong tumor killing effect under the alternating magnetic eld. Figure 7f shows the changes in animal weight after different treatments. There was no statistically signi cant difference between the 5 groups (p>0.05), indicating that the nanocarrier had no signi cant side effects.  Sequencing Figure 9 shows the sequencing results of the tumor tissues in the different groups. The results show that compared with the normal control group, the DOX-Fe 3 O 4 -PEG-PLA-NP group had 10 signi cantly differentially expressed RNAs and proteins, and more than half of the differentially expressed proteins were related to apoptosis. Abnormal expression of apoptosis-related molecules was also detected in the other nanocarrier groups. The molecular protein levels once again prove that the antitumor effects of these multifunctional magnetic nanocarriers is caused by apoptosis.

MRI results
In order to investigate the potential usefulness of the prepared MRI contrast agent, relaxivity measurements were carried out. Samples of two different SPIONs were prepared with different iron concentrations. Figure 10a shows the MRI results of the different materials, and as the iron concentration increased, the T 2 signal intensity gradually decreased. Figure 10b  magnetic resonance contrast agent has a higher relaxation rate. MRI of mice injected intravenously with different materials at different times (Figure 10d).

Discussion
In this study, we developed a new active ingredient characterization system, DOX-Fe 3 O 4 -PEG-PLA-NP, which is potentially effective and promising for cancer diagnosis and treatment. Polylactic acid (PLA) is a biodegradable synthetic polymer that has been approved by the US Food and Drug Administration (FDA) for medical applications [20] . However, due to its weak hydrophilicity, long degradation time and easy absorption by the liver and kidney, the applications of PLA are limited. In order to overcome these shortcomings, we coupled PEG with PLA to reduce absorption by the reticuloendothelial system [21] . In addition to the substance itself affecting the uptake by tumor cells, the size of the charge also affects the absorption capacity by tumor cells. Positively charged nanoparticles will be captured by the reticuloendothelial system (RES) and interact with negatively charged serum components, leading to further accumulation in organs such as the liver and kidney and a shortened blood circulation time. In addition, it has been proven that positively charged nanoparticles have a high a nity for negatively charged cell membranes and therefore have high inherent cell characteristics [22][23][24] . The PEG-PLA-NP material itself has a positive charge. In order to meet this challenge, considering that the external environment of tumor cells is more acidic (approximately pH 6.5) than the acidity of blood (pH 7.4), a novel PEG-PLA nanocarrier was designed. The carrier was designed to be ideal to stimulate system responsiveness, where these smart polymers could maintain a negative charge in blood circulation and minimize nonspeci c adsorption. After accumulation in a weakly acidic environment, they can spontaneously undergo a transformation from a negative to a positive charge, which promotes cell absorption or escape from lysosomes. Today, the use of magnetite nanoparticles has become a useful medical method for applications such as targeted drug delivery, MRI contrast agents [11] , magnetic hyperthermia [19] , cell separation, and DNA testing. In this study, we encapsulated Fe 3 O 4 and the tumorkilling chemical drug DOX into PEG-PLA nanocarriers, nally developing DOX-Fe 3 O 4 -PEG-PLA-NPs with tumor magnetic targeting, tumor imaging and tumor killing, which can be used as targeted macromolecules, imaging tags and a targeted drug delivery system.
As drug and magnetic material carriers, PEG-PLA nanomaterials display three properties: the ability to increase DOX solubility, a slow release effect and the active targeting to tumor tissues. DOX as a chemotherapy drug has two proposed mechanisms in cancer cells: 1) intercalation into DNA and disruption of topoisomerase-II-mediated DNA repair, and 2) production of free radicals, which disturbs the cellular membrane, DNA and proteins [25] . DOX is currently in clinical use in the form of doxorubicin hydrochloride because the original drug is insoluble in water, but its lack of tumor targeting limits its clinical applications [6,25] . PEG-PLA wraps DOX into nanoparticles, completely solving the problem of DOX solubility, which is one of the advantages of our PEG-PLA-NP drug delivery system. Another advantage of our nanocarrier system is its perfect slow release effects. The results of the drug release, pharmacokinetic, and drug distribution studies showed that the duration of DOX-Fe 3 O 4 -PEG-PLA-NPs in the body circulation (T 1/2 and peak arrival time) is much longer than that of the free DOX solution. In other words, at the same drug dose, nanocarriers can cause DOX to be more effective against tumors. The reasons affecting the slow release effects of the nanocarrier are as follows. 1) Tumor tissues proliferate rapidly, but the corresponding vascular dysplasia leads to the local acidic environment of the tumor [8,26] . The magnetic nanocarrier designed by our research group cleaves PEG-PLA in the acidic environment, thus releasing more drug. 2) The drug is encapsulated in the PEG-PLA nanocarrier, and the circulation of PEG-PLA in the blood weakens absorption by the reticuloendothelial system, so the drug showed reduced metabolism by the liver and kidney. A large amount of drug can therefore circulate in the body for a long time, allowing the drug to be slowly released into tumor tissues. The most important advantage of our nanodrug delivery system is its active targeting of tumor tissues. Fast-growing tumors require new blood vessels (neovascularization) or rerouting of the existing vessels adjacent to the tumors to provide enough oxygen and nutrition for their survival. This generates abnormal fenestrated endothelial structures around the tumors that are highly permeable for IONPs. These leaky vessels, which lack any associated lymphatic drainage, drive a unique process known as the enhanced permeability and retention (EPR) effect [27] . Therefore, the EPR effect may be one of the reasons that an increase in the distribution in tumor tissues and an increase tumor killing was observed. In addition, there are many factors that affect the distribution of nanocarriers in the body, such as the target organ type, the size of the nanocarrier, and the surface charge of the nanocarrier [10,28] . group was 2.79-fold greater than that in the free DOX group and 3.19-fold higher than that in the DOX-PEG-PLA-NP group after 2 h tail intravenous injection. Magnetic nanomaterials are driven to target positions using magnetic eld gradients. A recent study showed that using an external magnet around tumor sites signi cantly enhances the targeting ability of peptide-loaded IONPs and decreases liver uptake [29] . In this study, more magnetic nanocarriers brought chemotherapeutic drugs into the tumor tissue, which also demonstrated the tumor-targeting properties of the magnetic nanoparticles. In addition to the passive targeting of nanomaterial EPR, magnetic targeting may be another reason for the observed increase in drug distribution in tumor tissues. 2) Iron oxide generates reactive oxygen under the action of AMF, and the mechanism of this ROS killing tumor cells is very complex. It has been identi ed that various ROS forms in aqueous solution when magnetic nanoparticles are excited by alternating magnetic eld, such as hydroxyl radicals (·OH), hydrogen peroxide (H 2 O 2 ), superoxide radicals (·O 2 − ), and singlet oxygen ( 1 O 2 ). ROS acts on tumor cells and eventually leads to cell death by promoting apoptosis [11] . 3) Finally, under the action of AMF, the magnetic heat generated by the IONPs kills the tumor [30] . Hyperthermia, by de nition, is the application of heat above 40°C to kill tumor cells, which has been used alone and in combination with chemotherapy and radiation therapy. Higher temperatures (above 42°C) cause necrosis of living cells, denature enzymes, promote functional changes to DNA and RNA, and rupture cellular membranes, which releases cellular content, ultimately leading to cell injury and death [31] . In addition, increased heat increases the tumor killing effect of chemotherapeutic drugs.
The early diagnosis and treatment of cancer are key factors for good prognosis. Cancer diagnosis using nanotechnology is an emerging eld. Substantial efforts have been made in biomedical research to improve the sensitivity and accuracy of early detection methods to diagnose cancer and increase the effectiveness of treatment methods. Currently, the understanding of suitable biomarkers for imaging, the selection of imaging targets and contrast-enhancing materials, and the chemicals required for assembly of bioactive imaging probes is limited. In addition, there are many obstacles in the development of cancer-speci c imaging agents, such as 1) the delivery of probes to target tissues/tumors; 2) biocompatibility and toxicity; 3) stability of the probes and enhancement of effective signals in vivo; and 4) adequate imaging methods and strategies [17,18,32,33] . Nanoparticle drug delivery can provide more effective and less harmful solutions to overcome these problems. Fe 3 O 4 can enhance the sensitivity of MRI imaging. According to the quantum mechanical outer sphere theory, the T2 relativity or spin-spin relaxation is highly dependent on the saturation magnetization of the NPs. The greater T2 relativity is because larger NPs with higher saturation magnetization can afford more effective magnetic relaxations to the protons of water around the NPs [11,34] . The contrast effects of NPs are highly size-dependent. By controlling the size, we can achieve a much higher contrast enhancement. The particle size of the nanocarrier designed by our research group is above 100 nanometers, which can not only avoid oversized renal metabolism but also improve the imaging ability of ferric oxide. We used these four advantages of iron trioxide to improve the tumor killing and MRI imaging effects. The function of SPIONs in MRI contrast enhancement is attributed to the ability of SPIONs to change the nuclear spin relaxation of water protons and cause the target area to darken [35] . The advantages of magnetic resonance nanotechnology in measuring nanoparticle uptake come from the fact that the data collected are quantitative, have very effective soft tissue characterization and high tissue resolution, and have a tomography modality, which penetrates into the tissue without restriction. Therefore, as an effective MRI contrast agent, iron oxide nanoparticles have great potential in both imaging and image guidance. Speci cally, iron oxide nanoparticles can be detected with high sensitivity. In contrast to Gd, iron and polymer components have the advantages of biocompatibility, degradability and low toxicity. The degraded iron will be stored for further biological needs. Iron oxide nanoparticles are converted into elemental iron and nally incorporated into our body reserves or used to form hemoglobin [36] .

Synthesis of H 2 N-PEG-b-PLA nanoparticle micelles
With potassium bis(trimethylsilyl)amide (KHMDS) as the initiator, ethylene oxide and lactide were successively added to carry out anionic ring-opening polymerization, and the protecting group was removed by acid hydrolysis to produce the H 2 N-PEG-b-PLA block copolymer. Then, a certain proportion of dimethyl maleic anhydride was added to obtain HOOC-PEG-PLA.

Synthesis of DOX-PEG-PLA micelles
First, 500 µL of PEG 5k -PLA 5k (100 mg/mL), 1 mL of DOX (2.5 mg/mL) and 2.2 mL of chloroform were uniformly mixed and added into a mixture of 12 mL of 1% PVA and TPGS (external aqueous phase, PVA:TPGS 1:5). An oil-water emulsion was formed by using probe ultrasound for 5 minutes (80 W) in an ice bath. Finally, the mixture was added to 60 mL of 0.3% PVA (dispersed phase) and stirred overnight to volatilize the chloroform and solidify the PLA ball surface. A 100 kD ultra ltration tube was used for ultra ltration concentration cleaning. Finally, the sample was collected and stored at 4°C after a constant volume of pure water was added for a nal volume of 10 mL.

Preparation of DOX-Fe 3 O 4 -PEG-PLA micelles
First, 500 µL of PEG 5k -PLA 5k (100 mg/mL), 1 mL of DOX (2.5 mg/mL) and 2.2 mL of chloroform were uniformly mixed and added into a mixture of 12 mL of 1% PVA and TPGS (external aqueous phase, PVA:TPGS 1:5). An oil-water emulsion was formed by using probe ultrasound for 5 minutes (80 W) in an ice bath. Finally, the mixture was added to 60 mL of 0.3% PVA (dispersed phase) and stirred overnight to volatilize the chloroform and solidify the PLA ball surface. A 100 kD ultra ltration tube was used for ultra ltration concentration cleaning. Finally, the sample was collected and stored at 4°C after a constant volume of pure water was added for a nal volume of 10 mL. Encapsulation rate = W A /W B ×100% (1) Drug load = W A /W C ×100% (2) W A : DOX (mg) loaded into the PEG-PLA-FITC nanoparticles; W B : total dose of DOX (mg); and W C : total mass of the drug-loaded nanoparticles (mg).

Release study of nanocarriers in vitro
The release characteristics of DOX from nanocarriers were studied by the dialysis method. First, freezedried DOX-NPs and free DOX solutions (1 mg) were dissolved in DMSO (1 mL), transferred to dialysis bags (Snakeskin, Pierce Biotechnology, Rockford, IL, USA) (3500 kDa) and immersed in 50 mL of neutral saline (pH=7.4) or acidic saline (pH=6.5). The vials were shaken horizontally in a shaking water bath (100 rpm) at 37°C for 160 h. At predetermined time intervals, 5 mL from each saline sample was collected and replaced with an equal amount of fresh saline. Then, the DOX concentration in the physiological saline samples was analyzed by ultraviolet spectrophotometry to determine the cumulative DOX content released.
Cell culture and cytotoxicity measurements

Scratch experiment
First, two horizontal lines were drawn on the back of a 6-well plate. After the cells were digested, a cell suspension was made and plated, and when the con uence was above 90%, the cells were scratched with a 10 µL pipette tip on the bottom horizontal line, followed by gently rinsing with PBS 2-3 times, and replacing the medium with serum-free dosing medium for cell stimulation. Cultivation was continued in a 37°C, 5% CO 2 incubator. After 24 h, pictures were taken under a microscope, and the scratch area was calculated.

Flow cytometry
Flow cytometry was also utilized to examine the effects of the PEG-PLA-NPs on HeLa cell apoptosis.
Brie y, HeLa cells were    the data before and after enhancement will be measured and analyzed.

Supplementary Files
This is a list of supplementary les associated with this preprint. Click to download. gureS1.tif