extfieldsofstudy: Chemistry Medicine

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Materials Science
Chemistry

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Enhanced Synergism of Thermo-chemotherapy For Liver Cancer with Magnetothermally Responsive Nanocarriers

A combination of magnetic hyperthermia and magnetothermally-facilitated drug release system was developed as a promising strategy for liver cancer therapy. The thermosensitive copolymer, 6sPCL-b-P(MEO2MA-co-OEGMA) shows a good temperature-controlled drug release response. Mn-Zn ferrite magnetic nanoparticles (MZF-MNPs) exhibit a strong magnetic thermal effect with an alternating magnetic field (AMF). Owing to its high magnetic sensitivity, the magnetothermally-responsive nanocarrier/doxorubicin (MTRN/DOX) can be concentrated in the tumor site efficiently through magnetic targeting. Given this information, we synthesized MTRN/DOX which was composed of MZF-MNPs, thermosensitive copolymer drug carriers, and the chemotherapeutic drug---DOX, to study its anticancer effects both in vitro and in vivo. METHODS: MTRN/DOX was designed and prepared. Firstly, we investigated the accumulation effects of MTRN/DOX by Prussian blue staining, transmission electron microscopy (TEM), laser scanning confocal microscopy (LSCM) and conducted 7.0 T MRI. Following this, the magnetothermal effects of MTRN/DOX were studied using an infrared thermal camera. DOX uptake, distribution, and retention in tumor cells and the distribution of MTRN/DOX in vivo were then analyzed via LSCM, flow cytometry and live fluorescence imaging. Lastly, its anticancer effects were evaluated by MTT, AM/PI staining, Annexin-VFITC/PI staining and comparison of relative tumor volume. RESULTS: We found that MTRN/DOX can be efficiently concentrated in the tumor site through magnetic targeting, increasing the uptake of DOX by tumor cells, and prolonging the retention time of the drug within the tumors. MTRN/DOX showed good magnetothermal effects both in vitro and in vivo. Based on the above results, MTRN/DOX had significant anticancer effects. CONCLUSIONS: MTRN/DOX causes temporal-spatial synchronism of thermo-chemotherapy and together with chemotherapeutic drugs, produces a synergistic effect, which enhances the sensitivity of tumor cells to DOX and reduces their side effects.

Introduction
Chemotherapy is the major form of therapy for liver cancer treatment in addition to surgery, but has a number of limitations, which include low bioavailability of the drug and multiple side effects. Nanocarriers have been developed as novel drug carriers due to their attractive properties, including higher drug loading capacity, targeting and controlled release of drugs. Nanocarriers may increase the sensitivity of tumor cells, reduce the toxicity of chemotherapeutic drugs and allow these drugs to accumulate at the tumor site [1]. Currently, the most widely studied nanocarriers are polymeric Ivyspring International Publisher nanocarriers because of their high drug loading capacity and good stability [2,3].
As nanocarriers are responsive to certain stimuli, the drug carried by the nanocarriers can be released in response to a stimulus at the tumor site (controlled release), which reduces intensity of the side effects of the drug on other organs and improve its the bioavailability [4][5][6]. Thermosensitive copolymer nanocarriers trigger the release of the drug by altering the environmental temperature [7][8][9][10]. The structure of thermosensitive copolymers changes at different temperatures. The random copolymer composed of 2-(2-methoxyethoxy)ethyl methacrylate (MEO2MA) and oligo(ethylene glycol)methacrylate (OEGMA), collectively known as 6sPCL-b-P(MEO2MA-co-OEGMA) is a novel thermosensitive copolymer that uses temperature as a stimulating signal. It is a water-soluble, non-toxic, and non-ionic copolymer. By adjusting the ratio of the two monomers in the copolymer, the lower critical solution temperature (LCST), which is the critical point of phase transition [11], can be controlled at 43°C, a temperature at which the tumor cells are sensitized to chemotherapy. When the temperature reaches the LCST, the hydrophilic-hydrophobic transition will occur on the molecular chain of the copolymer, which allows achieving controlled release of the drug [10,12].
Numerous studies in recent years have shown that, compared to chemotherapy alone, thermo-chemotherapy possesses better effects [13][14][15][16][17][18]. The magnetic medium is introduced into the targeting tumor site, where it will produce heat by an appropriate frequency and intensity of an alternating magnetic field (AMF). Therefore, it can effectively achieve tumor hyperthermia [16,19]. Compared to the light-induced hyperthermia, the action of magnetic hyperthermia on tumor depth is not as limited [20,21]. In addition, as it is an efficient and easy-to-use chemotherapy sensitizer, magnetic hyperthermia may also sensitize the body to chemotherapy, allowing the achievement of an optimal combined therapy.
We used Mn-Zn-containing ferrite magnetic nanoparticles (MZF-MNPs) rather than traditional Fe3O4 particles. MZF belongs to the ferrous magnetic material family and possesses superior chemical stability and magnetic properties. MZF-MNPs can cause self-heating with AMF, and MZF can also serve as tumor imaging contrast agents in MRI [16]. In addition, previous experimental results have shown that nanoscale MZF has outstanding biocompatibility and minimal toxicity [22,23]. In terms of their physical structure, MZF-MNPs are spinel composite structures that have been subjected to high temperature sintering. Their internal structure is compact, thus allowing them to avoid from dissolution in strong acidic environments, which would otherwise create an excess of Mn 2+ , Zn 2+ , and Fe 3+ that could cause damage to tissues if they were to dissolve within the body [24].
A magnetic targeting drug delivery system (MTDS) can selectively target the tumor via a magnetic field, with a more powerful capacity for drug aggregation [25][26][27][28]. In the absence of targeting, the nanocarriers show specific tumor aggregation by means of enhanced permeability and retention (EPR) effects [29,30], but due to the low selectivity and specificity of passive targeting, their targeting is much less efficient. MZF possesses high sensitivity to its magnetic response. Therefore, it can be used as an ideal magnetic targeting material.
Based on the above considerations, we designed a magnetothermally responsive nanocarrier/ doxorubicin (MTRN/DOX) as a thermochemotherapeutic strategy for the treatment of liver cancer. MTRN/DOX contained the magnetic material MZF-MNPs, a thermosensitive copolymer drug carrier and DOX, which combined the magnetothermal effect of MZF-MNPs with the temperature-sensitivity of copolymer drug carriers. Magnetic targeting efficiently concentrates the nanocarrier at the tumor site, where AMF plays an important role in heating and allows controlled release of the drug at the tumor site to achieve spatial-temporal synchronism of thermochemotherapy. With this drug carrier system, we can improve the utilization of chemotherapeutic drugs and reduce their toxicity.

Synthesis and Preparation of MTRN/DOX
Ring-opening polymerization (ROP) and atom transfer radical polymerization (ATRP) were applied in preparing the thermosensitive amphiphilic blocked copolymer PCL-b-P(MEO2MA-co-OEGMA). LCST of the copolymer was precisely controlled by adjusting the ratio of MEO2MA and OEGMA.
(1) Synthesis of hydrophobic poly(ε-caprolactone) (PCL): We applied caprolactone (20.00 g), pentaerythritol (0.2390 g), and stannous octoate (175 μL) to a dry flask. After the flask was vacuumed, it was purged with argon. After repeating this process 3 times, the solution was left to react at 120°C for 24 hours under a magnetic stirrer. The product was then dissolved in methylene chloride. After being subject to precipitation for 3 times in ice-cold methanol, the reactants were dried under a vacuum until a constant weight was achieved.
(2) Synthesis of PCL-Br Macroinitiator: We weighed 0.866 g PCL and added it to a dry flask. We added methylene chloride (50 mL) and stirred until it dissolved and subsequently added 0.162 mL of triethylamine under an argon atmosphere to the flask. After the mixture was cooled to 0°C under a magnetic stirrer, 2-bromopropionyl bromide (0.122 mL) mixed in 20 mL dichloromethane was added dropwise over 20 minutes. The reaction was left under argon at room temperature for 48 hours. After completion of the reaction, the product was dissolved in dichloromethane. An equal volume of deionized water was added for extraction 3 times. The oil phase product was concentrated by rotary evaporation and was precipitated in ice-methanol for 3 times. Finally the product was dried under a vacuum until a constant weight was achieved.
(3) Synthesis of PCL-b-P(MEO2MA-co-OEGMA): We added 0.911 g PCL-Br macroinitiator, 2.820 g MEO2MA, 1.732 g OEGMA and 15 mL solvent tetrahydrofuran (THF) to a dried flask. The system was vacuum evacuated and subsequently purged with argon. We added 0.075 g of the ligand PMDETA and 0.058 g of the catalyst CuBr for 3 times. The reaction was left at 55°C for 5 hours. The product was dissolved in THF after reacting, and the copper salt was removed through a neutral alumina column. The obtained product was precipitated in ice-cold n-hexane and dried under a vacuum until a constant weight was achieved.
The MnxZn1-xFe2O4 was prepared by liquid phase thermal decomposition. The MnxZn1-xFe2O4, dried amphiphilic blocked copolymer PCL-b-P (MEO2MA-co-OEGMA) and DOX were dissolved in THF. The feed ratio of Mn0.6Zn0.4Fe2O4/amphiphilic blocked copolymer was controlled in a 1:1 ratio. After being treated in ultrasound for 15 minutes, the solution was poured into a dialysis bag (molecular weight: 8,000-14,000), and the water was changed every 6 hours. After 24 hours of dialysis, an aqueous solution of micelles was obtained.

Characterization and Properties of MTRN/DOX
(1) Transmission electron microscopy (TEM) (JEM-2010F, JEOL, Japan) was used to observe the morphology and size of the magnetic nanoparticles.
(2) A vibrating sample magnetometer was used to measure the hysteresis curve of MZF and MTRN/DOX at room temperature.
(4) MTRN/DOX solution was placed in a transparent vial with a magnet nearby in order to examine the magnetic feature of MTRN/DOX.
(5) A UV/Vis spectrophotometer (UV-Vis-NIR, Cary 5000, Agilent, USA) was used to measure the absorbance of the MTRN/DOX solution at 478 nm and the standard curve was plotted to calculate the drug loading content (DLC) of the micelles. DLC (%)=(mass of drug loaded in micelles/mass of drug loaded micelles)×100%.

Cell Culture and Tumor Modeling
Human hepatoma Huh-7 cells were cultured in Dulbecco's Modified Eagle's medium (DMEM) containing 10% fetal calf serum and 1% of the double-antibiotic (penicillin and streptomycin) in an incubator supplied with 5% CO2 at 37°C. Cells were passaged every 3-4 days. The cells at the logarithmic growth phase were suspended in PBS cells at a density of 1×10 7 cells/mL.
Male BALB/c mice were purchased from Shanghai Jiesijie Experimental Animal Co. Ltd. The 4-week-old nude mice were anesthetized with an intraperitoneal injection of 10% chloral hydrate, followed by slow injection of 0.3 mL of the Huh-7 cells suspension into the subcutaneous right hind legs. The mice were ready for experimentation when the tumors grew to a diameter of approximately 1 cm.

MTRN/DOX Accumulation in Tumor Tissues
Upon achieving 80% confluency, MTRN/DOX (100 μg/mL MTRN, 50 μg/mL MZF) was added to the Huh-7 cells. After undergoing incubation with the cells for 4 hours, the culture medium was removed, and the tumor cells were fixed with 4% paraformaldehyde. After being subjected to Prussian blue staining, the cells were observed under a microscope. Cells incubated with MTRN/DOX as previously described were digested in trypsin. After undergoing centrifugation, the supernatant was discarded, followed by the addition of a fixative (3% glutaraldehyde) to the cells. The cells were left at 4°C for 2 hours and converted into ultrathin sections for TEM. To observe the magnetic targeting effect of MTRN/DOX, we left the cells incubated with MTRN/DOX next to a magnet for 4 hours, followed by their subjection to Prussian blue staining as described above.
A 7.0 T small animal MRI (BioSpec 70/20, Bruker, Germany) was performed to observe the accumulation of MTRN/DOX in tumors in vivo. The mice were divided into 3 groups: a non-magnetic targeting group, a magnetic targeting group, and a control group that was injected with saline. Mice were anesthetized with 10% chloral hydrate via an intraperitoneal injection, followed by the slow injection of MTRN/DOX (1000 μg/mL MTRN, 500 μg/mL MZF) into the tail vein of the mice in the non-magnetic targeting group and magnetic targeting group. A magnet was placed on the tumor site of the magnetic targeting group mice for 4 hours after the MTRN/DOX injection. MRI T2-weighted imaging was performed prior to and at 24 hours after injection. MRI scan sequence parameters: T2-weighted (T2WI) spin-echo: TR=2635 ms; TE=33 ms; FOV=3×3 cm; Slice thickness=1 mm; Matrix=256×256; Scan time=4 min and 13 s.
The mice were killed after MRI imaging, and the tumor tissues were dissected and fixed, followed by Prussian blue and DAPI staining. The Prussian blue stained sections were observed under a microscope. Laser scanning confocal microscopy (LSCM) (TCS SP5, Leica, Germany) was used to observe the intracellular DOX fluorescence of DAPI staining sections. The fluorescence gray values of DOX were then measured to compare the quantitative DOX uptake in the tumor.

Magnetothermal Effects of MTRN/DOX
In vivo and in vitro magnetothermal effect experiments were carried out with AMF of f=114 kHz and Happlied=89.9 Ka/m. The MTRN/DOX (50 μg/mL MZF, 100 μg/mL MZF and 150 μg/mL MZF) and PBS as a control were subjected to AMF that was produced by a generator (SPG-20AB, Shuang Ping Tech. Ltd, China). The thermal images were recorded using an infrared thermal camera (RC05, Rinch, Hongkong China) and the temperature was also measured.
MTRN/DOX (500 μg/mL MZF) was injected into the tail vein of Huh-7 tumor-bearing nude mice. The mice were divided into 2 groups: a non-magnetic targeting group and a magnetic targeting group. The 2 groups of mice were subjected to AMF for 20 minutes at 24 hours after tail vein injection. An infrared thermal camera was used to observe and measure the thermal effects.
Huh-7 cells were incubated with free DOX and MTRN/DOX (150 μg/mL MZF, 15.78 μg/mL DOX) for 4 hours. The drug-containing medium was then removed and cells were washed with PBS. A portion of the cells was measured using flow cytometry (C6, BD, USA) based on DOX fluorescence; the rest of the cells were stained with DAPI, and LSCM was then used to observe the intracellular distribution of DOX and its relationship with the nucleus.
Next, the retention of intracellular MTRN/DOX and free DOX was measured. Cells were incubated with free DOX and MTRN/DOX for 4 hours. After being washed with PBS, the cells were grown in drug-free culture for 4 hours and cells were measured by flow cytometry based on their DOX fluorescence intensity. LSCM was applied to observe the intracellular distribution of DOX.
We studied the magnetothermal effects on cell uptake of the drug and the drug release. After the Huh-7 cells were incubated with MTRN/DOX (150 μg/mL MZF, 15.78 μg/mL DOX) for 2 hours, the cells were subjected to AMF of f=114 kHz, Happlied=89.9 Ka/m for 2 minutes. LSCM was used to observe the intracellular distribution of DOX and its relationship with the nucleus based on DOX fluorescence, and intracellular DOX fluorescence intensity was then measured via flow cytometry.

In Vivo Distribution of MTRN/DOX
Nude mice were divided into 3 groups: a non-magnetic targeting group, a magnetic targeting group, and a free DOX group. The mice were administered with MTRN/DOX or free DOX (DOX amount of 1000 μg/kg) via the tail vein. The mice in the magnetic targeting group were placed with a magnet on the tumor site for 4 hours and then at 4 hour, 24 hour and 48 hour time points following injection, the mice were killed to harvest their tumors, hearts, and kidneys.
The distribution of DOX in the tumors, hearts, and kidneys and their changes over time were imaged by live fluorescence imaging (Nightowl LB981, Berthold, Germany) and quantitatively compared by measuring the relative intensity of DOX fluorescence. A set of filter lens with excitation at 455 nm and emission at 560 nm was used.

In Vitro Hematological Analysis
Blood was obtained from healthy New Zealand rabbits and anticoagulated with potassium oxalate, at a final concentration of 1.0 mg/mL of blood. MTRN nanoparticles were rinsed three times with distilled water, ixiviated with 0.9% saline. The material detected was divided into four concentration groups, ie, 100, 200, 500, and 1000 μg/mL. 0.9% saline and distilled water were used as negative and positive controls, respectively. Each group contained three test tubes, each of which contained either 10 mL leaching liquor of MTRN, 0.9% saline, or distilled water. Then, 0.2 mL of diluted anticoagulated blood was added to each tube preheated for 30 minutes at 37°C. After incubation for 60 minutes at 37°C, the tubes were centrifugated at 2500 rpm for 5 minutes. Next, the supernatant fluid was assembled, and OD values were measured at 545 nm by UV-vis spectrophotometry. The hemolysisrate (HR) was calculated as follows: HR(%)=(OD of the experimental group-OD of negative control group)/(OD of the positive control group-OD of negative control group)×100%.

In Vitro and In Vivo Anticancer Studies
Huh-7 cells were seeded in 96-well cell culture plates, with each well consisting of 1×l0 4 cells, and then cultured in an incubator supplied with 5% CO2 at 37°C for 24 hours. Different concentrations of MTRN, MTRN/DOX, and free DOX were added to the 96-well cell culture plates and the cells were cultured for 48 hours. A group of cells was exposed to AMF with intensity f=114 kHz and Happlied=89.9 Ka/m for 2 minutes, and these were cultured for 24 hours. The cell viability of different treatments was calculated by the standard MTT assay.
With regards to AM/PI staining of the living/dead cells, the tumor cells in each group (MTRN, MTRN/DOX, MTRN+AMF and MTRN/DOX+AMF) were stained with a mixture of AM/PI, where AM was excited at 488 nm and PI was excited at 533 nm. The cells were observed under an inverted fluorescence microscope (Ti-S, Nikon, Japan) to distinguish between the dead and living cells.
The changes in cell apoptosis were investigated by staining with Annexin-VFITC/PI, and measured using flow cytometry. Huh-7 cells were seeded in culture dishes at a density of 1×10 5 cells/mL. The cells of each group (MTRN, MTRN/DOX, MTRN+AMF and MTRN/DOX+AMF) were trypsinized and stained with Annexin V-FITC and PI in the dark. Then, the cells were collected from each dish, followed by their subjection to flow cytometry for their fluorescence intensity, with the FL-1H channel detecting FITC at the wavelengths of 488 and 530 nm. The untreated Huh-7 cells were used as the negative control.
Tumor-bearing nude mice were observed until tumors grew to a diameter of 1 cm to start in vivo tumor suppression experiments. The nude mice were divided into nine groups (n=4 per group). The nude mice were anesthetized and then different treatments were administered intravenously (DOX dose of 1000 μg/kg, MTRN  certain groups of mice were exposed to AMF with intensity f=114 kHz, or site for 4 hours after injection of MTRN/DOX. Tumor sizes were monitored every 3 days for 18 days. The tumor volume was calculated: tumor volume V (mm 3 )=a (long diameter, mm)×b 2 (short diameter, mm 2 )/2. The relative tumor volume=V/V0, where V0 is the tumor volume at the start of treatment, V is the tumor volume after treatment. Mice body weights were measured at the start and end of treatment. The relative body weight=W/W0, where W0 is the body weight at the start of treatment.
The copolymer, 6sPCL-b-P(MEO2MA-co-OEGMA), prepared from 6sPCL and P(MEO2MA-co-OEGMA) ( Figure 1A) can be assembled into nanoscale micelles in PBS solution. Our previous studies showed that the LCST of 6sPCL-b-P(MEO2MA-co-OEGMA) was dependent on the ratio of MEO2MA to OEGMA [34]. When the ratio of MEO2MA and OEGMA was 92:8, the LCST was controlled at nearly 43°C, which was the chemotherapy sensitizing temperature for tumor cells. Therefore, we used 6sPCL-b-P(MEO2MA92%-co-OEGMA8%) copolymer micelles in our study.
Directed hyperthermia therapy of magnetic fluid by AMF has become a promising cancer treatment method. Compared to superparamagnetic iron oxide nanoparticles (SPIOs), the spinel structure composite of Mn-Zn iron oxide (MnxZn1-xFe2O4) has a higher saturated magnetization (MS) and stronger magnetic T2-weighted resonance effects [35,36]. The high specific absorption rate (SAR) of MnxZn1-xFe2O4 results in an excellent magnetothermal effect. It was reported that when x=0.6, the MS and SAR of Mn0.6Zn0.4Fe2O4 were maximized [37]. In order to achieve the best effects of hyperthermia and magnetic resonance imaging, we used Mn0.6Zn0.4Fe2O4 in our experiments.

Enrichment of MTRN/DOX on the Tumor Site
The MTRN/DOX and human hepatoma Huh-7 cells were mixed and incubated for 4 hours, followed by Prussian blue staining. As MTRN contains iron ions, it can be stained as blue particles by Prussian blue. Figure 2A shows the blue particles inside of the cells, indicating that the cell can engulf MTRN/DOX. TEM images show that ( Figure 2B, C) black granular MTRN/DOX are mainly contained in the cytoplasm.
In order to detect the in vitro magnetic targeting effects of MRTN/DOX, the cells were placed next to a magnet for 4 hours after MTRN/DOX were added. Figure 2D shows three points (Point I, II, and III) on the dish: Point I was on the edge of the magnet and Points II and III turned away from the magnet. Prussian blue staining showed that Point I formed an arc-like boundary along the edge of the magnet, with dense Prussian blue-stained particles inside of the magnetic field and decreased blue particles outside of the magnetic field. The blue particles on Point I formed an elongated arrangement towards the direction of the magnetic field ( Figure 2E). Points II and III showed fewer blue particles as the distance grew further away from the magnet ( Figure 2F, G). These findings suggest that MTRN/DOX could be used as the ideal magnetic targeting material. MZF-MNPs have a strong T2 relaxation effect and produce a low signal in MRI T2-weighted images. In order to evaluate MTRN/DOX for in vivo tumor aggregation and magnetic targeting, Huh-7 hepatoma-bearing BALB/c nude mice were used for in vivo MR imaging. 7.0 T MR imaging was performed before and 24 hours after injection into the tail vein. The T2WI image of nude mice in the 2 groups injected with MTRN/DOX following injection showed a flaky, patchy low signal area inside of the tumors, but larger areas and less intense signals were found within the tumors in nude mice of the magnetic targeting group, and the signals in the tumor of control group injected saline were not changed ( Figure 3A). Corresponding to MR imaging, there were more Prussian blue particles in tumor slices of the magnetic targeting group, in comparison to the tumor slices of the non-magnetic targeting group ( Figure 3B).   DOX emits red fluorescence at a wave length of 480 nm. Thus, intracellular red fluorescence can reflect the amount of DOX uptake of cells. Therefore, we used LSCM to observe the tumor slices. We found that the red fluorescence intensity in the magnetic targeting group was stronger than the intensity in the non-magnetic targeting group, and the red fluorescence gray values between the two groups had significant differences ( Figure 3C, D). This indirectly reflected the case of MTRN/DOX aggregation in the two groups.
The above results of MRI imaging, Prussian blue staining and LSCM observation revealed that MTRN/DOX could accumulate into the tumor tissues and in combination with the magnetic field, MTRN/DOX can be more efficiently targeted to the tumor tissue in vivo.

In vitro and In vivo Magnetothermal Effects of MTRN/DOX
MZF-MNPs in MTRN/DOX are a ferrous magnetic substance, so MTRN/DOX shows high SAR. With AMF, it can cause self-heating. As shown in Figure  4A and B, MTRN/DOX showed magnetothermal effects in a MZF content-dependent manner. When AMF (intensity f=114 kHz, Happlied=89.9 Ka/m) was applied for 1 minute, the temperature of MTRN/DOX (150 μg/mL MZF) in the tube increased by 5.5°C (from 25.0°C to 30.5°C). When applied for 3 minutes, the temperature increased by 18°C, reaching 43.0°C of LCST. In 5 minutes, it reached 51°C, and in 10 minutes it reached 60.5°C. With a lessening of MZF content, the magnetothermal effects of MTRN/DOX gradually lowered. As a control, the PBS temperature remained basically unchanged. Based on the initial calefactive velocity of MTRN/DOX, the maximum SAR of MTRN/DOX is 905.6 W/g, corresponding to the highest Happlied of 89.9 kA/m. MTRN/DOX had excellent magnetothermal effects in vitro. We then studied the magnetothermal effects of MTRN/DOX in vivo. Nude mice were divided into magnetic targeting and non-magnetic targeting groups. After subjecting the mice to injection of MTRN/DOX (150 μg/mL MZF) into the tail vein, the 2 groups of mice were placed under the same intensity of AMF, followed by observation of their effects using the infrared thermal imager. Without AMF, the surface temperature of the tumor in both groups of nude mice was approximately 33°C. The temperature of the tumor in the mice with AMF was found to be higher than in those without AMF. For 10 to 15 minutes with AMF, the heating curve of both groups reached a plateau. For 20 minutes, magnetic targeting group reached 51°C whereas the non-magnetic targeting group reached 43°C only ( Figure 4C, D). Compared to the previous findings, a greater proportion of MTRN/DOX accumulated on the tumor site in the nude mice of magnetic targeting group.
Therefore, they showed advanced magnetothermal effects on the tumor site. It should be noted that only the temperature of the tumor surface was measured, and the temperature inside the tumor should be higher. In addition, we found that the thermal effects were mainly found in the tumor region, and no significant heating was found in other parts of the body. These findings suggested that if we applied thermal therapy, it would not affect other organs and tissues. Therefore, MTRN/DOX demonstrated significant thermal effects in the tumor in vivo, and the surface temperature of the tumor in the magnetic targeting group reached over 50.0°C, which could cause irreversible damage to tumor cells [38,39].

Uptake, Distribution, and Retention of DOX in Tumor Cells
DOX is an anthracycline anticancer drug. DOX has a wide anti-tumor spectrum and premium efficacy [40]. However, due to its severe toxicity, long-term use of DOX can lead to dose-dependency, irreversible lesions in the heart and kidney, and bone marrow suppression. In order to conquer these challenges, researchers have been investigating an effective way to reduce the clinical toxicity and improve the treatment efficacy. Currently, nano-drug carrier systems are considered to be effective.
We first examined the release rate of MTRN/DOX. As seen in Figure 5A and B, at 37°C, the release rate of MTRN/DOX (150 μg/mL MZF) was very low for 10 minutes. At 5 hours later, the release rate reached approximately 20%, followed by a flattened plateau curve up to 24 hours, then remained at approximately 22%. This indicated that in the absence of AMF, the release of DOX in MTRN/DOX was very slow and limited. We then observed the effects with AMF ( Figure 5A). At a temperature of 25°C in the MTRN/DOX (150 μg/mL MZF) solution, for 3 minutes with AMF, the cumulative release rate of DOX from MTRN/DOX was 1.1%. After 5 minutes, it was 13.5%, and for 10 minutes it was 41.3%. MTRN/DOX shows magnetothermal effects in a MZF content-dependent manner, so the release rate of MTRN/DOX is also dependent on the MZF content. Figure 5A indicates that the speed of DOX release with AMF showed a positive correlation with the MZF content.
Compared to the previous heating curve of MTRN/DOX (150 μg/mL MZF), we found that for the first 3 minutes with AMF, the DOX release curve was gradual, but after 3 minutes, the slope of the release curve steepened and the release rate increased ( Figure 5E). This is because for 3 minutes with AMF, the temperature of the MTRN/DOX (150 μg/mL MZF) solution reached 43.0°C, which was the LCST of the thermosensitive copolymer. At this temperature, the 6sPCL-b-P(MEO2MA-co-OEGMA) chain inside the micelle shell became hydrophobic, and the core-shell structure changed, which resulted in the rapid release of DOX wrapped inside the core. Accordingly, the slope of the release curve of MTRN/DOX (50 μg/mL MZF) and MTRN/DOX (100 μg/mL MZF) increased at the 6th and 4th minute after exposure to AMF ( Figure 5C, D). At the two time points, the temperature of MTRN/DOX (100 μg/mL MZF) and MTRN/DOX (50 μg/mL MZF) was approximately 43.0°C.
Huh-7 cells were incubated in the medium containing MTRN/DOX and free DOX, respectively at 37°C for 4 hours. Cells incubated with MTRN/DOX showed stronger red fluorescence intensity by LSCM and flow cytometry ( Figure 6A), and the red fluorescence was mainly in the cytoplasm. Cells incubated with free DOX displayed the red fluorescence mainly within the nucleus ( Figure 6A). Free DOX primarily acts on the DNA, so it directly enters the nucleus. As seen in Figure 5A and B, little DOX was released from MTRN/DOX during the short period of time at 37°C, and as seen in Figure 2 B and C, the MTRN/DOX particles that the cells engulfed were mainly located in the cytoplasm. As a result, little red fluorescence was found in the nucleus of cells incubated with MTRN/DOX. This indicated that, without AMF, little DOX was released from MTRN/DOX and entered the nucleus for killing of tumor cells. Consequently, limited DOX toxic effects would occur in tissues that do not heat up.
In order to detect the retention time of DOX in the cells, we incubated the cells with MTRN/DOX or free DOX for 4 hours, then removed them from incubation. After 4 hours, we found that the cells incubated with MTRN/DOX still showed strong red fluorescence, whereas the cells incubated with free DOX showed weak fluorescence. Flow cytometry showed similar results ( Figure 6B).
In conclusion, we discovered that even without AMF, MTRN/DOX could promote tumor cell uptake with encapsulated DOX, and DOX of MTRN/DOX retention in tumor cells was significantly prolonged. We detected the magnetothermal effects on cell uptake ( Figure 6C). Compared to the cells that were not exposed to AMF, cells with AMF showed increased intracellular DOX fluorescence intensity, and we found that the red fluorescence was partly distributed in the nucleus. This was due to the high temperature caused by AMF destructed cell membrane stability, which increased the permeability and membrane fluidity, thus making the drug enter tumor cells easily [41]. Conversely, when the temperature reached the LSCT of the thermosensitive copolymer, DOX release from MTRN/DOX was accelerated and entered the nucleus. Thus, MTRN/DOX with AMF not only increased the uptake of DOX in tumor cells, but also promoted DOX release, which synchronized thermotherapy and chemotherapy inside the cells. In this manner, chemotherapy was largely enhanced.

In Vivo Distribution of MTRN/DOX
Based on DOX auto-fluorescence, we studied DOX distribution in the nude mice by dissected organ imaging. We were able to find the differences between the distribution and duration of DOX in the tumors, hearts, and kidneys by observing and measuring the fluorescence of DOX. As in Figure 7, 4 hours after tail vein injection, tumor tissues from the free DOX group showed specific tumor fluorescence intensity. The fluorescence intensity rapidly reduced at 24 hours, and the DOX fluorescence was very weak at 48 hours. The kidneys from the free DOX group showed strong fluorescence at the corresponding 4 hours after tail vein injection. The fluorescence intensity gradually weakened the kidneys over time. This showed that free DOX can be rapidly distributed and cleared by the body, and may not act on the tumor for a long time. The DOX fluorescence intensity in tumors of the non-magnetic targeting group gradually increased over time, and a certain intensity of fluorescence still existed after 48 hours. This phenomenon is related to EPR. Compared to normal tissues, tumor tissues are rich in blood vessels and have a wider blood vessel gap, poor structural integrity, and lack lymphatic drainage, which is responsible for their selective high permeability and retention of macromolecular substances.
This effect makes nanocarrier macromolecules accumulate in the tumor lesions more easily by passive dispersion, where they play a role in passive targeting. This effect is relatively slow, but once inside the tumor site, the nanocarrier may be retained for a long time and maintain a high concentration of DOX in the tumor. Compared to the non-magnetic targeting group, at 4 hours, the magnetic targeting group displayed fairly high intensity fluorescence within the tumor due to the dual effects of magnetic attraction and EPR. The fluorescence intensity within the tumor was higher at each time point in the magnetic targeting group than in the non-magnetic targeting group. Compared to the free DOX group, mice of the magnetic targeting group and non-magnetic targeting group showed a lower distribution of fluorescence within the kidney. There was slower DOX excretion in these 2 groups and DOX concentration can be maintained for a long time in the body (Figure 7).
Cardiotoxicity is one of the serious adverse reactions of DOX. We compared cardiac DOX distribution at different time points. At each time point, the fluorescence intensity in the heart of both the two MTRN/DOX groups was found to be lower than the intensity of the free DOX group. Moreover, the fluorescence intensity of the heart from the magnetic targeting group was found to be weaker than the intensity of the non-magnetic targeting group (Figure 7). These findings showed that MTRN/DOX can reduce cardiotoxicity caused by the free DOX.
Overall, these results revealed that MTRN could promote DOX targeted accumulation in tumor tissues with an external magnetic field, increased DOX uptake of tumor cells, and prolonged the drug retention time in the tumor, while it reduced the distribution of DOX in the hearts and kidneys and thus reduced the side effects of the drug.

Cytotoxicity and Anticancer Efficacy of MTRN and MTRN/DOX
MTRN is injected intravenously, so it is essential to investigate its hemo-compatibility. The percentage of hemolysis caused by MTRN at different concentrations was depicted in Figure 8A. For all test samples, the hemolysis rates were less than the international standard (5%). This clearly suggests that MTRN have excellent hemo-compatibility when directly contacted with blood.
An MTT assay was used to measure the cytotoxicity of MTRN and MTRN/DOX. As in Figure  8B, when Huh-7 cells were incubated with MTRN with a concentration from 10 μg/ml to 1000 μg/ml for 48 hours of incubation, the cell viability was above 90%. This finding suggested good biocompatibility of MTRN with no cell-killing effects. We then studied the cytotoxicity of MTRN and MTRN/DOX (MTRN concentration was 9.5 g/mL, 19 μg/mL, 47.5 μg/mL, 95 μg/mL and 380 μg/mL; the corresponding DOX concentration was 0.5 μg/mL, 1.0 μg/mL, 2.5 μg/mL, 5.0 μg/mL, and 20 μg/mL) without AMF, and the same concentration of free DOX was used as the control. After 48 hours, we found that without AMF, cell activity was inhibited to some extend with MTRN/DOX, but at the same DOX concentration, MTRN/DOX without AMF showed less cell-killing capacity than the free DOX group ( Figure 8C). Based on the studies described above, we believe that the low drug release rate without AMF led to its relatively weak cytotoxicity.
With AMF, as shown in Figure 8D, the MTRN and MTRN/DOX groups showed significantly increased cytotoxicity and significantly reduced cell viability, whereas the cytotoxicity in the free DOX group presented no significant changes. By AM-PI live (green) and dead (red) staining, we directly visualized the cell-killing effects of MTRN, MTRN+AMF, MTRN/DOX, and MTRN/DOX+AMF (at a MTRN concentration of 380 μg/mL). As shown in Figure 9A, the MTRN/ DOX+AMF group showed mostly dead cells stained red, followed by the MTRN+AMF and MTRN/DOX groups with many dead cells, but the MTRN group showed substantially more green-stained live cells. Cell apoptosis was then quantitatively studied by staining with Annexin-V-FITC/PI and subjecting them to flow cytometry. In the same manner as AM-PI staining, the proportions of cells in early and later apoptosis (Q4+Q2) from high to low were MTRN/DOX+AMF (80.7%), MTRN+AMF (63.9%), MTRN/DOX (54.2%) and MTRN (3.7%) ( Figure 9B).  These results are due to the low thermal resistance of tumor cells. Moreover, heat promoted the uptake and the release of the drug and increased tumor cell sensitivity to chemotherapy, which together improved the anti-tumor effects.
We also evaluated the in vivo therapeutic effects of MTRN/DOX on tumors. Depending on the temperature of the thermal effects, thermotherapy was divided into warm thermotherapy (40-43°C) and high temperature thermotherapy (43-70°C). Warm thermotherapy was applied for the whole body, and high temperature thermotherapy was used for the topical treatment of tumors. The previous experimental results showed that with AMF for 15 minutes, magnetic targeting of MTRN/DOX increased the tumor surface temperatures by up to 50°C, and the internal temperature was higher than 50°C. In addition, the high temperature was confined within the tumor. Therefore, it belonged to the local high temperature therapy. When the temperature was maintained at 50-52°C for only 4-6 minutes, it would be required for the induction of tumor cell necrosis [42].
Nude mice bearing tumors were divided into 9 groups according to different treatment modalities:  Figure 10A, B and C). After 18 days of treatment, group IX showed the best treatment effects: a relative tumor volume reduction by approximately 75% due to the aggregation of the magnetic targeting and the synergistic effect of thermo-chemotherapy. Group VIII also showed certain effects with a tumor reduction of approximately 20%. Additionally, group VII showed slower tumor growth. The tumor volume increased by approximately 2 times, due to MTRN-produced heat with AMF, which showed certain inhibition of tumor growth. Other groups showed rapid tumor volume increments. Tumor volumes in groups I, II, III, V, and VI enlarged up to 7 times compared to the original volume. In contrast to the in vitro study, which showed less cytotoxicity in MTRN/DOX than in free DOX, the in vivo anti-tumor effect was found to be better in MTRN/DOX than in free DOX. This may be due to the rapid clearance, short retention, and lower tumor uptake of free DOX in vivo. According to previous results, owing to the EPR effect in tumor tissues, MTRN/DOX can partially concentrate in the tumor tissues, and the release rate of MTRN/DOX was low, so that it can induce a long sustained action in the tumor. Therefore, the in vivo anti-tumor efficacy of MTRN/DOX was better than the efficacy of free DOX.
At the end of the 18-day treatment, compared with group I (PBS), the relative body weights of the nanocarrier-treated groups (III, IV, VII, VIII and IX) were not decreased and were actually increased (groups IV, VII, VIII and IX) ( Figure 10D). It also indicates that both MTRN and MTRN/DOX have outstanding biocompatibility with minor toxicity and significant therapeutic effects.

Conclusions
In summary, we designed and prepared a novel magnetothermally-responsive nanocarrier system with outstanding biocompatibility. This system synchronized the magnetothermal therapy and drug release; magnetic targeting and synergy of thermo-chemotherapy effectively enhanced the sensitivity of tumor cells to existing chemotherapy drugs and reduced their side effects. Our results showed that MTRN, as a novel nanocarrier in synergy with thermo-chemotherapy, has enormous potential in liver cancer therapy.


text:
A systematic analysis of the expression of the anti-HIV VRC01 antibody in Pichia pastoris through signal peptide optimization

Pichia pastoris (Komagataella phaffi) has been used for recombinant protein production for over 30 years with over 5000 proteins reported to date. However, yields of antibody are generally low. We have evaluated the effect of secretion signal peptides on the production of a broadly neutralizing antibody (VRC01) to increase yield. Eleven different signal peptides, including the murine IgG1 signal peptide, were combinatorially evaluated for their effect on antibody titer. Strains using different combinations of signal peptides were identified that secreted approximately 2–7 fold higher levels of VRC01 than the previous best secretor, with the highest yield of 6.50 mg L−1 in shake flask expression. Interestingly it was determined that the highest yields were achieved when the murine IgG1 signal peptide was fused to the light chain, with several different signal peptides leading to high yield when fused to the heavy chain. Finally, we have evaluated the effect of using a 2A signal peptide to create a bicistronic vector in the attempt to reduce burden and increase transformation efficiency, but found it to give reduced yields compared to using two independent vectors.

Introduction
The broadly neutralizing antibody (bNAb) VRC01 is currently undergoing Phase II clinical trials to determine its effectiveness in preventing HIV infection in high risk women and in men and transgender people who have sex with men [1,2]. VRC01 works by binding to the gp120 envelope glycoprotein and blocking interaction with the CD4 + receptor; thus, preventing viral entry into the T-cell [3]. In vitro VRC01 has been shown to neutralize 91% of HIV isolates, and therefore is a promising candidate in the fight against HIV [4]. Results from two initial Phase I clinical trials indicate that VRC01 has been identified as a being able to delay the return of viral replication following interruption of antiretroviral therapy [5].
In most cases VRC01 is produced in human embryonic kidney (HEK) cells, although production in plants has also been reported [6]. Yields for plant production were up to 100 mg/kg in stably expressing lines. We have previously reported on the production of the bNAb VRC01 using Pichia (syn. Komagataella) pastoris, with yields up to 3.05 mg L −1 in shake flasks [7]. Our research utilized the murine IgG1 signal peptide (GWSCIILFLVATATGVHSQ) for the first time in P. pastoris, which outperformed the more popular α-mating factor from Saccharomyces cerevisiae [8,9]. P. pastoris is often chosen as an expression host due to the high cell densities that can be achieved, up to 130 g L −1 dry cell weight, and therefore the corresponding high volumetric productivities [10]. P. pastoris secretes very few native proteins, thus purification can be made easier by directing recombinant proteins to the supernatant through the use of a signal peptide. There are many reported instances where the choice of signal peptides impacts the yields achieved for expression; for instance He et al. found that utilizing a bovine-casein signal peptide for the secretion of the xlyanase xynB resulted in lower yields than using the α-mating factor (α-MF) pre-pro peptide from S. cerevisiae [11]. Gasser et al. were unable to secrete antibody fragments using a mAb leader signal peptide, but were able to secrete the same products using the α-MF [12]. Conversely the expression of bovine pancreatic trypsin inhibitor (BPTI) was unsuccessful when using the α-MF, but utilizing the human serum albumin (HSA) signal peptide resulted in successful secretion into the medium [13].
There are various truncations to the α-MF signal peptides that have been demonstrated to increase the cleavage efficiency of the STE13 protease for some proteins [14][15][16]. In addition, a series of other signal peptides have in various forms successfully resulted in the production of recombinant proteins; α-amylase [17], glucoamylase [18], inulinase [19] invertase [20], killer protein [19], lysozyme [21], and human serum albumin [13,22]. As the optimal choice of signal peptides is often protein specific, testing different signal peptides should influence overall yield.
Another method to improve yield has been by using bicistronic vectors, which in the case of antibodies results in the production of both the heavy and the light chain from a single vector. These can be generated using internal ribosome entry sites (IRES) [23] or through the use of 2A peptide sequences [24]. Previously antibody fragments have been expressed using two expression cassettes in tandem; however, this requires two promoter and transcription terminator regions [12]. The IRES and 2A both utilise a single promoter, reducing the size of the vector and increasing transformation efficiency, as well as potentially reducing the impact of loop out recombination [25,26]. IRES are large sequences and preferentially express the upstream protein at a 10-fold higher level of expression than the downstream protein, which explains their decreasing popularity [27]. The 2A sequence functions by causing a ribosome skip that cleaves between the terminal glycine and the beginning of the final protein resulting in two separate polypeptides [28][29][30]. 2A sequences have been used in P. pastoris for the production of complex multi-enzyme pathways [31] as well as for the production of antibodies [32]. The original 2A sequence was discovered in foot-andmouth disease virus (FMDV), F2A, in 1991 [33]. In addition, there are the Thosea asigna virus sequence (T2A) and the porcine teschovirus-1 sequence (P2A) [24]. The P2A has been used for the production of antibodies in a streamlined system outlined by Shah et al. [32]. All three variants have been analysed in P. pastoris [31], and Geier et al. identified that using the F2A can result in production of the fusion protein as opposed to discrete protein. Furthermore, work carried out in mouse lines indicates expression of the second protein is higher when using the T2A compared to the P2A [34].
Here we set out to increase the production of the bNAb VRC01 by exploring a combinatorial library of signal peptides, including the murine signal peptide first described in our previous paper. Furthermore, we evaluated the impact of using a 2A signal peptide to produce both chains of the antibody from a single bicistronic vector.
Pichia pastoris (syn. Komagataella phaffi) strain Δku70 (CBS 12694, CBS-KNAW, Fungal Biodiversity Centre, Utretcht, The Netherlands) was used as the background strain. For cloning into P. pastoris 5-10 μg of plasmid DNA was linearized with PmeI at a single restriction site within the AOX1 promoter. The vectors were transformed into cells by electroporation according to recommendations in the Pichia Expression manual (Thermo Fisher Scientific) and grown for 3-5 days at 30°C on YPD medium containing both 100 μg mL −1 Zeocin (Thermo Fisher Scientific) and 100 μg mL −1 Nourseothricin (Jena Bioscience) to select for double transformants. For nomenclature purposes the heavy chain will be denoted as (H) and light chain as (L) following the signal peptide, i.e. αKH-GAL would be the combination of the α-mating factor K signal peptide fused to the heavy chain and the glucoamylase signal peptide fused to the light chain.
For the bicistronic vectors the T2A and light chain was amplified from r_αA-VRC01 and integrated into the pPICZ-heavy vector, in the orientation of light-T2A-heavy [36]. After transformation into P. pastoris selection was carried out on YPD plates containing 100 μg mL −1 Zeocin (Thermo Fisher Scientific).

Copy number analysis
Genomic DNA was extracted using the DNeasy ® Plant Mini Prep Kit (Qiagen, Crawley, UK), quantified by Nanodrop™ (Thermo Fisher Scientific) and normalized to 0.5 ng μL −1 using distilled H 2 O. Quantitative PCR was run on genomic DNA using KICStart ® SYBR ® Green qPCR ReadyMix™ (Sigma Aldrich) in an Eppendorf Mastercycler ® ep realplex quantitative cycler (Eppendorf UK Ltd, Histon, UK). The known 1 copy clone α-1 was used as a reference strain and all strains were compared accordingly [7]. Data was analysed using the Pfaffl method, based on ΔΔ-Ct [37,38] and normalized to ACT1 as the housekeeping gene. Primers for the heavy chain were AAT CAC AAG CCC AGC AAC AC and GGG CAT GTG TGA GTT TTG TCA C resulting in a 74 bp amplicon. Primers for the light chain were TGT CTT CAT CTT CCC GCC ATC and ATT CAG CAG GCA CAC AAC AG resulting in a 70 bp amplicon. Primers for ACT1 were GCT TTG TTC CAC CCA TCT GT and TGC ATA CGC TCA GCA ATA CC resulting in a 163 bp amplicon. Cycling conditions were 95°C for 5 min followed by 40 cycles of 95°C for 5 s, 58°C for 15 s and 72°C for 10 s with a melting curve afterwards to ensure a single product was being measured. Error bars are representative of the Gaussian error propagation of the standard deviation of s

Protein expression
Expression screening in P. pastoris was performed in 24 deep-well plates in 3 mL of medium and sealed with Breathe-Easy ® sealing membrane (Sigma Aldrich). Larger scale expression in P. pastoris was performed in 250 mL glass baffled flasks in 25 mL of BMGY medium. Cells were incubated at 30°C, 216 rpm for 24 h in BMGY to allow growth before being centrifuged at 4000 rpm for 5 min. The supernatant was removed and the medium replaced with BMMY to induce expression. Cultures were left to express at 20°C, 216 rpm for 72 h before being harvested. Every 24 h the culture was supplemented with 0.5% (v/v) methanol.

Enzyme-linked immunosorbent assay (ELISA)
ELISA was performed on neat supernatant cultures from flask expression. For the total Ig ELISA, Nunc Maxisorp (Thermo Fisher Scientific) high-binding 96-well microplates were coated with 100 μL anti-human Kappa and Lambda light chain mix (Southern Biotech, Birmingham, US) in PBS at 4°C overnight. Wells were washed with 0.05% (v/v) Tween in PBS four times, and subsequently blocked with 200 μL 0.05% (v/v) Tween and 1% (v/v) Bovine Serum Albumin (BSA) in PBS (assay buffer) for 1 h at 37°C. To generate a standard curve 50 μL standard antibody in 5-fold serial dilution was added to triplicate wells, with a starting concentration of 1000 ng mL −1 . The plates were then incubated for 1 h at 37°C, washed, and 100 μL detection antibody added (anti-human IgG HRP; Southern Biotech), diluted to 1:10,000 in assay buffer. After 1 h incubation at 37°C wells were washed a further 4 times. 50 μL TMB (KPL Inc, Milford, USA) was added and incubated for 5 min 50 μL of Stop solution (KPL Inc) was added and the absorbance measured at 450 nm immediately. Some ELISAs included a biotin amplification step where the detection antibody used was an anti-human IgG Biotin labelled (Southern Biotech), diluted to 1:10,000 in assay buffer, followed by a wash step and then incubation with a streptavidin-HRP conjugate (R&D Systems), diluted 1:200 in assay buffer, before washing and development of the signal with TMB as detailed above. To determine antigen-specific binding wells were coated with 50 μL CN54-gp140 antigen at 1 μg mL −1 in PBS at 4°C in place of the IgG capture antibodies. The antibody concentration reported by the gp140 ELISA method is lower than that of the total antibody method due to different binding affinities to the plate bound antigen. Yields of VRC01 for shake flasks were recorded previously [7].

Expression of VRC01 using the paired signal peptide
The signal peptides from the ATUM SM expression library vectors were amplified and cloned into the existing pPICZ-heavy or pJAN-light vectors [7]. This created 20 vectors, containing the 10 signal peptides with either the light or heavy chains in addition to the original vectors containing the murine signal peptide (Fig. 1). Of the new 10 vectors, each vector pair containing the same signal peptide (e.g. SAH and SAL) on the heavy and light chain was transformed in Δku70 with selection on both 100 μg mL −1 Zeocin and 100 μg mL −1 cloNAT. This background strain was chosen as it prevents off-target recombination, and as both vectors integrate into the same locus ensures correct integration [41]. The presence of both the heavy and light chain sequences was confirmed by colony PCR as previously described (data not shown) [7].
Emulating the screening process that is used in industry, we randomly selected four independent clones to test for expression levels [42,43]. Clones were expressed in microtiter plates and the expression was determined by ELISA (Fig. 2) and Western blot ( Fig. 3 and Supplementary Fig. 1). In our original paper, the most highly expressed strain was M2, which in small scale produced 2.95 μg L −1 , which is represented on Fig. 2 with the red line. None of the strains with new signal peptides outperformed this strain in small scale expression (Fig. 2). However, there was noticeable variation in expression of the heavy and light chains when culture supernatants were analyzed by Western blot, as indicated in the representative Western blots shown in Supplementary Fig. 1. For example, using the GA signal peptide resulted in an abundance of light chain, but limited amounts of heavy chain, with most being retained in the pellet (Supplementary Fig. 1). On the other hand, using the SA signal peptide resulted in higher levels of expression of the heavy chain than in GA, although there is still evidence that the heavy chain was not fully secreted ( Supplementary  Fig. 1). The ratio of light to heavy chain is more balanced when using the SA signal peptide, and this is represented in the ELISA results (Fig. 2) where there is increased signal using the SA signal peptide compared to the GA signal peptide.
Western blot heavy and light chain bands were measured by densitometry and calculated as a ratio to the previously published M2 strain (Fig. 3). Light chains were highly secreted in strains with the αK, GA, IV, LZ and SA signal peptides, although IV shows proportionally higher levels of light chain maintained within the pellet than the other strains. Conversely the αKS, αT and KP signal peptides resulted in very low levels of light chain secretion, with the former two showing also very little retained within the pellet (Fig. 3A). For the heavy chain, the AA, αK, LZ and SA signal peptides show the highest levels of secretion, although all also show antibody being retained within the pellet. Levels of antibody in strains using the αKS are low, but this could be due to the low levels of light chain production in these strains. αT shows higher levels of heavy chain in the supernatant than is retained in the pellet, and this implies that the low total antibody yield observed for this strain (Fig. 2) is not as a result of the heavy chain being maintained within the pellet, but lower light chain expression, suggesting that this signal peptide is not compatible with high levels of production of VRC01.

Expression of VRC01 using combination signal peptides
The Western blot results ( Fig. 3 and Supplementary Fig. 1) facilitated selection of signal peptides for further study. It is apparent that the choice of signal peptides affects the efficiency of secretion of heavy and light chains and therefore, using a combination of different signal peptides could increase yield. To select which signal peptides to explore in our combinatorial library, we used different criteria for the heavy and light chain. It has been shown that light chain can be secreted independently of the heavy chain, but not the other way around [12,44,45]. Therefore, signal peptides from strains that showed high levels of light chain in the supernatant where chosen for further strain construction irrespective of the secretion of the heavy chain. For the heavy chain, signal peptides were chosen based on either a high level of expression in the supernatant from the ELISA data (e.g. SA, LZ) or those that showed low levels of expression of the light chain and evidence of heavy chain accumulation in the cell pellet (e.g. AA, KP) since the antibody secretion in these strains could have been negatively impacted by low availability of the light chain. As a result, the following signal peptides were chosen to be fused with the heavy chain: AA, αK, αKS, IV, KP, LZ and SA. For the light chain the αK, GA, LZ and SA signal peptides were chosen. Furthermore, as the murine signal peptide resulted in the highest levels of secretion in our previous study, this was also used for both the heavy and the light chains. Therefore, we constructed a combinatorial library of 36 different sets of strains by transforming each combination of heavy and light chain vector into P. pastoris.
Four colonies from each combination were selected and antibody was expressed in microtiter plates as previously described. Antibody yield was analysed by ELISA (Fig. 4) and Western blots (data not shown). Compared to the previously reported strain that used the murine secretion signal for both heavy and light chain (indicated by the red line), there were strains with higher yield, unlike when using identical signal peptides from the library for both the heavy and the light chains. Interestingly, the murine signal peptide appeared to be the best fusion partner for the light chain in all cases. As we have analysed multiple clones in order to account for clonal variation [42,43] it was of note that all clones of AAH-ML, LZH-ML and SAH-ML combination outperformed the M2 strain in small scale expression, suggesting that these signal peptides result in efficient processing of the antibody for secretion. The best secreting clones of AAH-ML, LZH-ML and SAH-ML produced 1.5, 5 and 7 fold higher titers than M2, respectively.
The LZH-ML and SAH-ML strains had the highest antibody expression levels overall. Therefore, we scaled up expression of these strains to shake flasks and compared the titers of total and gp140 specific antibody to those from M2 (Fig. 5). Despite the small-scale expression results, not all of the clones outcompete M2 in antibody yield in shake flask expression. It has been reported previously that reproducible scale up is difficult to achieve in P. pastoris, often due to the high oxygen levels required, which can result in cell death [46]. Nonetheless compared to M2, two clones of each of the strains produced higher total and gp140 specific antibody concentrations (LZHML1, LZHML4, SAHML2 and SAHML4).
As our initial selection of clones was random, we undertook an investigation to understand if the differences in yield observed from different strains were solely a result of differences in gene copy number [43]. In the industrial selection of clones, companies will often screen thousands of colonies based on titer alone, and only assess integration sites and copy number after a good candidate has been identified [47]. Therefore, from our best secreting clones we analysed the copy number of the four LZHML and SAHML strains to ensure that the differences we had measured for titer were not purely as a result of copy number differences. We had previously determined that the M2 strain has two copies of both of the heavy and the light chain. Interestingly, the lower expressing clones of LZHML and SAHML had higher copy numbers, ranging from 4 to 8 copies of the heavy chain and 3 to 6 copies of the light chain ( Supplementary Fig. 2). This is consistent with previous findings that implied the benefits of increasing gene dosage plateau at a maximum value where secretion saturation can occur, resulting in detrimental effects on yield on further increase in copy number [26]. In fact, there was a negative correlation between the number of heavy chain copies and the level of expression (r = 0.715), which was less pronounced for the light chain correlation (r = 0.5951). This is supported by the findings that the heavy chain cannot be secreted without the light chain; therefore the heavy chain remains the limiting factor for the concentration of intact antibody [12,44,45].
From the best secreting strains LZHML1, SAHML2 and SAHML4 had one copy of heavy chain and two copies of light chain whilst LZHML4 had two copies of each the heavy and the light chains (comparable to the M2 strain analysed). This indicates that the variation observed between the levels of expression using different signal peptides is not due to differences in copy number, but results from the different signal peptides.
Whilst there is no significant difference when comparing all of the strains, SAHML4 had a total expression of 6.5 mg L −1 and 187 μg L −1 of gp140 specific antibody. This is a 2.13 and 1.77 fold increase respectively compared to M2, which was the highest secretor found from our previous experiments. Even though our yields are still comparatively low compared to those reported for other types of proteins expressed in P. pastoris [10], they are on par with that previously reported for expression of other antibodies. For example, Shah et al. reported yields of 100-500 μg L −1 for antibody production in microtiter plates and scFvs titers of up to 3.5 mg L −1 were reported in shake flasks (in a mutS strain KM71) [32,48,49]. Optimising growth conditions and using bioreactors may result in further improved yield. Furthermore, as the VRC01 gene used was not codon optimized, this may have limited productivity, although codon optimization does not always result in improved yields [50].

Expression of VRC01 using the T2A signal peptide
A recent investigation into creating an automated pipeline for the  production of HIV-specific monoclonal antibodies in P. pastoris utilized a 2A peptide sequence to generate a single vector to express both the heavy and the light chains [32]. We decided to generate bicistronic vectors for the MHML, LZHML and SAHML combinations as these gave the highest yields when utilising separate plasmids. As the T2A sequence from Thosea asigna virus was shown to produce discrete proteins in a previous investigation and results in higher expression of the second protein, this peptide sequence was used as a linker between the light and heavy chains [31,34]. A single bicistronic vector should reduce the burden on P. pastoris and increase transformation efficiency [25,51]. It has previously been demonstrated that an excess of light chain can promote folding and increase antibody stability; therefore the light chain was placed upstream of the T2A signal peptide [32,52].
Initially, twenty clones from each of the transformations were expressed in microtiter plates and the yield observed via dot blot (data not shown). From the dot blot results, we selected four of the highest expressing clones, which were expressed in shake flasks for 72 h in the methanol containing medium BMMY (Fig. 6). Despite the reduced burden, no clones showed higher expression than the M2 strain originally described. The MLT2AMH strains had a mean total antibody expression of 958 μg L −1 and a mean of 36.8 μg L −1 of gp140 specific antibody, both over a third lower than the M2 strain with the same signal peptides. The highest yield came from the MLT2SAH strains; however, the mean expression was still lower at 1.7 mg L −1 and 38.2 μg L −1 for total antibody and gp140 specific antibody concentrations respectively. The highest individual expressing strain was MLT2ASAH1 at 2.9 mg L −1 of total antibody, although for gp140 specific antibody this was MLT2ASAH4 at 77.7 μg L −1 . We previously reported that the signal from the gp140-specific ELISA was between 4 and 5% that of the total antibody ELISA for antibody expressed in mammalian 293T cells as well as P. pastoris [7]. This difference is due to different binding affinities of the antibody to the plate bound antigen, in contrast to the much higher affinity capture ELISA used to detect total IgG. The plate bound antigen may be distorted due to electrostatic stresses, leading to a reduction in the ability of the antibody to bind. However, here we see variations in this percentage depending on the signal peptide used, with ranges between 1.76% and 4.3%. Since some strains show percentages in the same range as our previous measurements, this disparity is unlikely to be due to incomplete processing of the 2A sequence and is perhaps more likely to be signal peptide related (Supplementary Fig. 3).
It would appear from our findings that the use of a peptide cleavage signal was not effective for increasing the expression of the VRC01 antibody. Further investigations are required to determine if this is specific for T2A or whether other 2A peptides may result in higher yields, or whether placing the heavy chain upstream of the light chain results in increased production.

Conclusions
Through the combinatorial exploration of signal peptides, we have increased the expression of the bNAb VRC01 in P. pastoris by 2-7 fold with yields of up to 6.5 mg L −1 in shake flask expression. This was through the combination of different signal peptides on both the heavy and the light chain. We have determined that the use of the murine IgG1 signal peptide resulted in the highest yields when fused to the light chain, with greater flexibility on the signal peptide for the heavy chain. Surprisingly the use of the T2A peptide sequence resulted in lower yields than when using independent plasmids. Further investigations into the choice of a cleavage sequence would be required to determine whether this is the most efficient approach for antibody   6. Concentration of total and gp140 specific antibody of MLT2ALZH, MLT2SAH and MLT2AMH. Antibody concentrations were calculated of total IgG1 and gp140 specific antibody. Samples were expressed in BMMY for 72 h in 25 mL cultures. M is representative of previous results reported from large scale expression [7]. A) Total antibody concentration. B) gp140 specific antibody concentration. The error bars are representative of the standard deviation from four independent clones. production in P. pastoris.

Author contributions
RA designed and performed the experiments and helped draft the manuscript. PM performed the ELISA experiments and analysis. RS, PM and KP helped design the experiments and draft the manuscript. RS and KP conceived the study. All authors read and approved the final manuscript.

Declarations of interest
None.


extfieldsofstudy: Medicine Biology

s2fieldsofstudy: Biology Medicine

text:
No effect of thymosin beta‐4 on the expression of the transcription factor Islet‐1 in the adult murine heart

Abstract The transcription factor Islet‐1 marks a progenitor cell population of the second heart field during cardiogenesis. In the adult heart Islet‐1 expression is limited to the sinoatrial node, the ventricular outflow tract, and parasympathetic ganglia. The regenerative effect in the injured mouse ventricle of thymosin beta‐4 (TB4), a 43‐aminoacid peptide, was associated with increased Islet‐1 immunostaining, suggesting the induction of an Islet‐1‐positive progenitor state by TB4. Here we aimed to reassess this effect in a genetic model. Mice from the reporter mouse line Isl1‐nLacZ were primed with TB4 and subsequently underwent myocardial infarction. Islet‐1 expression was assessed 2, 7, and 14 days after infarction. We detected only a single Islet‐1+ cell in 8 TB4 treated and infarcted hearts which located outside of the sinoatrial node, the outflow tract or cardiac ganglia (in ~2500 sections). Two cells were identified in 5 control infarcted hearts. TB4 did not induce LacZ positivity in ventricular explants cultures of Isl1‐nLacZ mice nor did it affect the density of LacZ+ cells in explant cultures of nLacZ+ regions of the heart. In summary, we found no evidence that TB4 reactivates Islet‐1 expression in adult mouse ventricle.

| INTRODUCTION
Regenerative strategies might represent new therapeutic approaches to heart failure, which is still the leading cause of death in the western world. 1 One possible treatment strategy aims at stimulating proliferation and differentiation of endogenous progenitor cells in the adult heart. Although several endogenous progenitor cell populations have been described, their exact physiological role and cardiogenic potential is still unknown or controversial. 2 Islet-1 + cells are a prominent example among the cells in question. During cardiac development Islet-1 marks a progenitor cell population of the second heart field. These Islet-1 + cells are capable of differentiating to cardiomyocytes, smooth muscle cells, and endothelial cells and contribute to the right ventricle, the right atrium and the outflow tract. [3][4][5] Islet-1 knock-out leads to severe cardiac malformation, 6 substantiating its crucial role for cardiac development. In contrast, Islet-1 expression in the adult heart is limited to very distinct regions 7,8 : cells of the outflow tract and parasympathetic ganglia as well as the sinoatrial node, for which an important functional role of Islet-1 has recently been established. [9][10][11][12] Thymosin beta-4 (TB4) is a 43-aminoacid peptide which derives its name from the fact that it was first discovered in thymus. It is, however, expressed ubiquitously and present in most cells at high concentration. 13 Initial studies focused on its role as an actin-sequestering molecule that inhibits the polymerization of globular actin (Gactin). 14 More recently pleiotropic effects of TB4 have been described. Its role in cardiac development is controversial. While some reported that TB4 is essential for cardiac development and promoted cell survival, migration, and regeneration after cardiac injury, 15,16 others failed to observe a cardiac phenotype in TB4 knockout mice, 17,18 suggesting that it is not required for cardiac development. Since the initial description of its beneficial properties in myocardial injury 15 several studies have evaluated TB4 in the context of myocardial injury. TB4 improved vasculogenesis, reduced infarct size, prevented ventricular rupture and improved left ventricular function after myocardial infarction. It also extended the time window for cardiac regeneration after birth and amplified the effect of Gata4, Mef2c, and Tbx5 to transdifferentiate fibroblasts to cardiomyocytes. [19][20][21][22][23][24][25][26] Administration of TB4 before myocardial injury activated epicardial progenitor cells (marked by the expression of Wilm's tumor 1 protein, WT1) and thereby led to de novo cardiomyogenesis. 27 In this work, the epicardial progenitor cells in the infarct area stained positive for Islet-1 shortly after injury. Given the limited specificity of immunolabeling in histological preparations we set out to reevaluate the effect of TB4 on distribution and role of Islet-1 + cells after injury using a genetically defined model.

| Animals
The investigation conforms to the guide for the care and use of laboratory animals. This study was carried out in accordance with the recommendations of the NIH (Publication No. 85-23, revised 1985).
The protocol was approved by the local authorities (Beh€ orde f€ ur Gesundheit und Verbraucherschutz-Veterin€ arwesen/Lebensmittelsicherheit). Isl1-nLacZ mice were generated and characterized as described previously. 3 Investigations were done in male and female heterozygous Isl1-nLacZ mice on a mixed Black Swiss and C57BL/6J background.

| Experimental myocardial infarction
Myocardial infarction was induced by permanent ligation of the left anterior descending artery (LAD) as previously described. 28 Mice were isoflurane-anesthesized (2.5%) and mechanically ventilated.
Animals were assured to a warming platform in a supine position.
Buprenorphine (0.05 mg/kg body 86 weight) and carprofen (5 mg/kg body weight) were injected subcutaneously 15 minute prior to the operation. Left-lateral thoracotomy was performed. The pericardium was opened and the LAD was ligated with a single stitch (8-0 Prolene suture, Ethicon, Norderstedt, Germany). Analgetic treatment (buprenorphine 0.05 mg/kg body weight twice per day) and carprofen (5 mg/kg body weight/day) was continued 5 days postoperatively. Injection of TB4 (12 mg/kg bodyweight) was performed following a protocol from Smart et al. 27 TB4 was injected daily for 7 days prior to LAD-ligation and every other day after the surgery.
Control animals were injected with PBS. Mice were sacrificed 2, 7, and 14 days after LAD-ligation by cervical dislocation. Hearts were then perfusion fixed with 4%formaldehyde in situ, harvested and washed in 0.9% NaCl.

| Histology
Hearts were embedded in a sucrose (30%)/Tissue-Tek (Sakura) mixture (1:1) and serial cryosections (10 lm) of the heart were acquired in a transversal (short) axis. All sections were collected on Superfrost Plus-slides (Fisher Scientific). Hematoxylin and eosin staining (H&E) was performed to allow for an overview and Picro-Sirius red staining was performed to analyze infarct size. Consecutive sections were used for immunofluorescence staining. X-Gal + structures were identified with an x10 objective.
These structures were further analyzed in higher magnification with an x40 objective. Cells showing a clear blue nuclear signal and a costaining with DRAQ5 were counted (nLacZ + ). Pictures were taken with an Axio Scope 2 microscope driven by the Axiovison software (Zeiss).
Immunostaining was performed at 4°C over night for the first anti-

| Infarct size measurement
Sections from infarcted hearts were stained with Picrosirius red and examined with a Zeiss Axiovert 25 microscope using a x1.25 objective. Images were captured with a Jenoptik ProgRes SpeedXT core 5 camera driven by the ProgRes Capture Pro software. Ima-geJ 1.8.0 software was used to measure scar and left ventricular myocardial areas. The scar was measured by an investigator who was blinded to the identity of the sections. Infarct scar area and total area of the left ventricular myocardium from 7 to 8 sections per heart were traced manually and measured automatically.

| TB4 effects in vivo
We first analyzed the effect of TB4 on the healthy myocardium in Isl1-nLacZ (n = 3) and WT (n = 3) animals. Mice were treated with TB4 (12 mg/kg body weight, i.p.) over the course of 7 days. TB4 treatment had no effect on body weight, and the animals showed no obvious alteration in behavior (Figure 1). Although no nLacZ + cells could be detected after treatment with TB4 in WT animals as expected, all Isl1-nLacZ animals showed nLacZ + cells in the 3 established distinct structures: the sinoatrial node, the outflow tract and We then evaluated the effect of TB4 on Islet-1 expression after myocardial injury. LAD ligation was performed after 1 week of daily TB4 administration (12 mg/kg body weight per injection).
Following LAD-ligation TB4 was injected every other day. Hearts were harvested 2 (control n = 1; TB4 n = 2), 7 (control n = 3; TB4 n = 4) and 14 days (control n = 1; TB4 n = 2) after myocardial infarction ( Figure 3A). Infarct size was small in TB4 primed hearts and control animals (4.74 AE 0.99% in PBS treated animals vs. 5.58 AE 1.38% in TB4 primed mice). Altogether, approximately 4000 tissue sections were analyzed for nLacZ + structures. Again, we found positive cells in the sinoatrial node, the outflow tract, and cardiac ganglia. We identified 3 nLacZ + cells that could not be ascribed to any of these 3 structures ( Figure 3B): a single cell was detected in a TB4-treated Isl1-nLacZ mouse 14 days after LAD-ligation. This cell was localized in an epicardial tissue adhesion. Costaining with DRAQ5 revealed a nuclear localization of the LacZ-signal. Adjacent to that cell, a second blue signal suggested another nLacZ-positive structure. However, this structure did not stain positive for DRAQ5 ( Figure 3B). The other 2 nLacZ + cells were detected in control-injected Isl-nLacZ mice 7 days after infarction ( Figure 3B). DRAQ5 confirmed a nuclear localization of the LacZ-signal in both cells. We initially considered more cells in the infarct region to be nLacZ + . However, a more rigorous evaluation revealed that those cells either showed a cytosolic LacZ-staining pattern ( Figure 4A and B) or were located in the outflow tract ( Figure 4C and D).

| TB4 effects in cardiac explant cultured
It has been reported that TB4 upregulates Islet-1 in explant cultures from the heart and eventually leads to the differentiation of epicardial cells to cardiomyocytes in vitro. 29 We therefore also investigated the effect of TB4 on outgrowth cultures of heart pieces from Isl1-nLacZ mice. Unexpectedly, we initially observed a high density of nLacZ + cells, irrespective of the supplementation of TB4 in these explant cultures (n = 3 per group). To further understand the origin of these cells, we separated the heart in an Islet-1 + basal part and an Islet-1 À apical part and incubated pieces of them separately (n = 3 per group). While explant cultures from the basal part of the heart reliably contained nLacZ + cells, explant cultures derived from the apical part never did (5A-H), irrespective of TB4 treatment.
Immunofluorescence labeling revealed that the nLacZ + cells expressed smooth muscle actin (SMA) and discoidin domain-containing receptor 2 (DDR2, Figure 5I and J). Dissection of the outflow tract area prior culture resulted in cell cultures that did not contain nLacZ + cells in the basal part ( Figure 5L, n = 2). Explant cultures derived from the outflow tract in contrast resulted in nLacZ + cells ( Figure 5K).

| DISCUSSION
This study evaluated the effect of TB4 on the expression of the transcription factor Islet-1 in a well-characterized animal model that allows for an easy and unambiguous identification of Islet-1 + cells.
We identified only 1 single nLacZ + cell outside of the already These cells expressed the fibroblast markers SMA and DDR2. Our model does not allow lineage tracing and we therefore cannot rule out that these cells are (partially) derived from an Islet-1 À population. The fact that nLacZ + cells occurred only in explant cultures from the basal part of the heart indicates that these originated from preexisting Islet-1 + smooth muscle cells and fibroblasts that we have previously described in the outflow tract. 7 This assumption is further supported by the fact that dissection of the outflow tract prior to culture led to nLacZ À cell cultures. The negative findings of this study are in line with results from other groups resulting in a controversial discussion of the role of TB4 in the heart. 17,18,[30][31][32][33][34] Interestingly there is conflict on its role during development as well as on its therapeutic potential. Our study focused on the expression of Islet-1 after TB4 treatment and was not designed to evaluate de novo cardiomyogenesis or the therapeutic potential of TB4 in general. In particular the number of animals used in our study is too small to draw conclusions about functional effects or infarct size.
Yet, our study is in agreement with a recent publication that did not detect an increase in Nkx2.5-positive cells after TB4 treatment in injured hearts. 34 The fact that we did not detect nLacZ + cells in the ventricle supports our previous findings and suggests that possible regenerative effects of TB4 in the injured heart are independent of reexpression of the transcription factor Islet-1.

ACKNOWLEDG EMENTS
We thank RegeneRx Biopharmaceuticals Inc, Rockville, MD for providing Thymosin Beta 4, S. Evans for providing Isl-1-nLacZ mice and L. Carrier for the maintenance of the Isl-1-nLacZ mouse line.