Efficient Synergistic Immunotherapy for Inhibiting Tumor Postoperative Recurrence and Metastasis via Calcium Alginate Hydrogel Nanosystem

: Immunotherapy is expected to become an promising strategy in inhibiting tumor postoperative recurrence and metastasis. However, the effect is still unsatisfactory because of lacking cooperativity between various therapeutic methods. In this study, we designed an efficient synergistic immunotherapy system as an all-around and multi-dimension method for inhibiting tumor postoperative recurrence and metastasis. The efficient synergy lay in enhancing immune effect and eliminating immune suppression in the meantime by took advantages of CpG oligodeoxynucleotides (CpG ODNs) and antiPDL1 antibody. We introduced nanomaterials based on the calcium alginate hydrogel which has an acknowledged biological safety to implement the above strategy and make the system multifunctional. This nanosysterm have both therapy function and long-term monitoring capability in vivo to evaluate the recurrence and metastasis situation of postoperative residual tumor cells conveniently in real time. In vitro and vivo results proved that that this system could achieve a much better tumor inhibition efficiency and a good monitoring effect. This efficient synergistic immunotherapy nanosystem is expected to become a new promising strategy for postoperative immunotherapy of tumor.


Background
Surgical resection is the most common choice to eliminate tumors in clinical at present [1,2]. Postoperative recurrence and metastases often occurs along with it [3][4][5][6]. Therefore many people have aimed on develop strategies to prevent cancer recurrence and metastasis after surgery. Traditional methods such as chemotherapy or radiotherapy are often used to assist postoperative treatment [7][8][9]. But the effects of these traditional methods is still very limited and there are many defects of them include toxic side effects as well as drug resistance and so on. The failure of effectively inhibiting postoperative recurrence and metastasis is still the main reason make tumors difficult to cure. There is an urgent need to develop a long-term, systemic and high-security therapeutic strategy to solve this problem. In recent years, tumor immunotherapy has become a hot spot in the field of tumor therapy especially after surgery and the recent research has shown that using immunotherapy methods to assist surgical resection will become a promising strategy to inhibit tumor recurrence and metastasis in the postoperative treatment of tumors [10][11][12][13]. There are many available immunotherapy methods have been put into application at present [14,15].
However, the effect is still unsatisfactory because of lacking cooperativity between various therapeutic methods so there is a urgent need to develop a synergistic strategy to obtain mutually reinforcing and enhanced therapeutic effect.
In this study, we designed an efficient synergistic immunotherapy system as an all-around and multi-dimension method for inhibiting tumor postoperative recurrence and metastasis. The efficient synergy lies in enhancing immune effect and eliminating immune suppression in the meantime take advantage of CpG ODNs and antiPDL1 antibody. First, CpG ODNs is a single-stranded oligonucleotide fragment containing specific non-methylated CpG dinucleotide sequences [16][17][18][19]. It's a generally recognized Toll-like receptors (TLRs) agonists in recent years [20]. TLRs are expressed by most antigen presenting cells (APCs) such as dendritic cells (DCs), which is the most important class of APCs in the body. TLRs on DCs can sense a wide range of danger signals, which are conducive to the maturation of DCs. It is also contribute to stimulate the secretion of related cytokines and the maturation of cytotoxic T lymphocytes which can enhance the anticancer activity of various cancer treatment [21]. Second, immune checkpoint inhibitors have shown encouraging results in the treatment of various cancers [22]. In particular the antiPDL1 antibody, which is a key pathway to relieve the tumor inhibition of immune response [23][24][25].
This efficient synergistic immunotherapy system can promote the induction of persistent and specific anti-tumor T cell effects in vivo.
In recent years, nanomaterials have received wide attention in tumor immunotherapy because of its high efficiency drug delivery ability and multifunctional integration capability [26][27][28]. Therefore we introduced nanomaterials to implement the above strategy and gave the system more functionality. For inhibiting recurrence in situ, it was hoped that a smaller amount of CpG ODNs could produce sufficient immune effects. So we used the high biocompatibility injectable calcium alginate (CA) hydrogel to load CpG ODNs [29,30]. Through the sustained release effect of hydrogel could improve the immune activation effect of CpG ODNs. Furthermore, we also loaded the quantum dots-gold nanoparticles (QDs-AuNPs) probe based on the fluorescence resonance energy transfer (FRET) theory in the gel which can detect the content of a tumor marker carcinoembryonic antigen (CEA) in vivo [31]. CEA content reflected the level of tumor recurrence, so the tumor recurrence could be monitored in real time through the fluorescence signal of the probe which fixed in the tumor site. Then we used the same material to prepare calcium alginate nanoparticles as a carrier of antiPDL1 antibody which could improve the treatment effect rely on the advantages of nanomaterials to assist orthotopic therapy and inhibit the tumor metastasis. Meanwhile, ICG was loaded as imaging agent to diagnose tumor metastasis and drug aggregation. This nanosystem based on the calcium alginate hydrogels have both therapy function and long-term monitoring capability in vivo which could be injected into the body after surgical resection to inhibit tumor recurrence and metastasis. In the meanwhile, it could evaluate the recurrence and metastasis situation of postoperative residual tumor cells and the treatment effect conveniently in real time through the imaging testing.
A series of in vitro and in vivo experiments were conducted to test the effect of this nanosystem. We expected this efficient synergistic immunotherapy nanosystem could provide a new promising strategy for the postoperative immunotherapy of tumor.

Calcium alginate hydrogel loads the CpG ODNs to enhance the immune effects through sustained release function.
First we aimed at using a sustained release strategy to attain a stronger immune effects by only adding a small quantity of CpG ODNs. We chose injectable calcium alginate hydrogel as the carrier to achieve our purpose which have a widely use and high biocompatibility. Sodium alginate and CaCO3 were used to prepare the calcium alginate hydrogel, gluconolactone (GDL) was used to dissociate the Ca 2+ from CaCO3 then cross-linked with sodium alginate slowly (Figure 2a and Figure S1). It was liquid at first and will became solid after a while so it's a injectable hydrogel which is convenient to use ( Figure S2). In order to acquire the suited calcium alginate hydrogel with long time fixation and sustained drug release function, we explored the preparation formula of our hydrogel based on the 2.5 % sodium alginate which is the commonly used percentage (Figure 2c). The percentage of CaCO3 and GDL determine the solidification time of hydrogel. But if the percentage of GDL is too high, the PH of hydrogel will be too acidic to damage the tissue. After our exploration 8 % CaCO3 and 8 % GDL were chose to prepare the hydrogel for next use and it had a relatively higher biosafety for cells ( Figure S3). And use this preparation formulathe the hydrogel will become solid in about 5 minutes which is opportune enough to inject into the body and fix in situ rapidly. Then the CpG ODNs was loaded in the hydrogel and the release rate was test in a simulated physiological environment (37 ℃, PBS). It was proved that the CpG ODNs can sustained release in 24 hours (Figure 2d).
The quantity of CpG ODNs we used (20 μg) was fewer then it be used alone in previous research (50 μg) because of the sustained release function of the hydrogel.
We tested the therapeutic effect of our CA hydrogel loaded the small quantity CpG ODNs in vivo (Figure 2e). The ELISA measurement of immune cytokines showed that our design can produce more stronger immune effect then used the CpG ODNs alone at the same condition ( Figure 2f). The difference of the tumors growth between different groups also proves the CpG ODNs loaded in the alcium alginate hydrogel can obtain the enhanced therapeutic effect in inhibiting tumor postoperative recurrence through its sustained release function ( Figure 2g) and had no effect on the health of the mice ( Figure S5). The treatment principle was shown in Figure 2b.

CEA probe fixed by hydrogel in situ for monitoring the tumor recurrence.
Base on the therapy of CpG loaded in the CA hydrogel, we conceived of introducing a mechanism which is simple and easy to implement for monitoring the therapy result and the tumor recurrence situation in real time in vivo. To achieve this purpose we constructed the QDs-AuNPs probe based on the FRET theory ( Figure 3a).
The DNA aptamer1 and aptamer2 can conjugate to QDs and AuNPs respectively. The CEA aptamer on the one hand could connect the QDs and AuNPs then bring them closer together to achieve the FRET effect because of its two matched ends with aptamer1 and aptamer2 respectively. On the other hand the middle of the CEA aptamer could bond to CEA specifically so the FRET effect will disappear when there is any CEA exists.  (Figure 3g and 3h). We also verified the biosecurity of the probe ( Figure S7). Then we loaded the probe in the hydrogel we prepared before and tested the detection efficacy. The fluorescence signal of the probe reflects the level of CEA content ( Figure 3i). In sum, our recurrence monitoring mechanism showed a hopeful ability for further application and is expected to be a new detecting tool in vivo.

CANPs carried antiPDL1 antibody to assist orthotopic therapy for inhibiting
the tumor recurrence and metastasis to achieve efficient synergistic immunotherapy.
Except the recurrence of tumor often occurs after surgery, the metastasis along with it also makes tumor difficult to cure. In order to assist orthotopic therapy and inhibit the tumor metastasis all over the body, we designed another nanosystem used calcium alginate hydrogel too. The synthesis method was shown in figure 4a. At first we prepared calcium alginate nanoparticals (CANPs) used sodium alginate and CaCl2.
In the meantime we added ICG as an imaging agent in the preparation process. The ICG@CANPs have a uniform morphology (size are around 110 nm) and a good dispersion in water (Figure 4c). The good biosecurity is also be verified for further applying in vivo (Figure 4d). The CANPs is a good carrier because of there are plenty of carboxyl on it so it is easy to proceed surface modifying such as the reaction between carboxyl and amino which we used to conjugate the antiPDL1 antibody. In  (Figure 5e and 5f). The inhibition effect of metastatic tumour in lung was displayed by pictures of lung tissue H&E stain sections (Figure 5g). All of the above results showed that our efficient synergistic immunotherapy nanosystem could acquire a better therapeutic effect then other control groups and had no effect on the health of the mice (Figure S8 and S9). In particular it was more efficacious than only activating immune effect or only eliminated immune suppression which is often used previously in clinical.

Discussion
In this study, an efficient synergistic immunotherapy nanosystem based on

Preparation of injectable calcium alginate hydrogel
The preparation method of injectable calcium alginate hydrogel is referred to the previous methods. First prepared the 2.5 % sodium alginate (SA) solution in reserve.
Then added appropriate amount of CaCO3 powder into the SA solution above mentioned to make the mass fraction of CaCO3 is 4 %, 6 % and 8 % respectively.
Finally added the GDL which compound is 4 % -16 % respectively. Mixed the compound sufficient and it solidified after different times in different ratio.

Synthesis of the QDs and the AuNPs
The water-soluble and biocompatible glutathione-coated CdTe QDs was prepared by previous method with slight modification. Prepared 50 mL CdCl2·2H2O

Preparation of the CEA probe
The prepared ODs are rich in carboxyl because of the GSH on the surface.
Activated the carboxyl for concatenating the aptamer1 with amidogen Utilized the sulfydryl on the AuNPs to conjugated the aptamer2 with sulfydryl (CTGGTATAAAA-SH 3'). The prepared AuNPs were washed up with deionized water then dissolved in 2% SDS solution (PH≈3). Take 2 mL AuNPs (5 mg/mL) in the tube. Added 10×TBE and NaCl maked the ultimately concentration was 1×TBE and 500 mM. Then added 10 μL aptamer2 which have been reducted with TCEP before.
Shook slowly for 5 min at room temperature. Then washed three times with deionized water.

Preparation of the nanosysterm①
Take 100 μL above probe and added appropriate amount of SA and CaCO3 maked the concentration was 2.5 % and 0.8 % respectively. Then added 20 μg CpG ODNs and homogeneous mixed. At last added the GDL which concentration is 8 % finally.

Preparation of CANPs
Partially hydrolyzed the sodium alginate according to traditional method before used. Added 3 mL HCl (3 M) in 100 mL 1 % sodium alginate solution and heated up to 50 ℃ with reflux for 20 min. The product was centrifuged and collect the sediment.

Preparation of the nanosysterm②
Added 5 mg ICG in 5 mL CANPs solution (10 mg/mL) and stirred for 8 h.
Washed with deionized water and collected the sediment. Used NHS and EDC activated the carboxyl of CANPs. Added 100 μg antiPDL1 antibody in the above CANPs and stirred for 12 h.

Cell culture and imaging
The 4T1 cell lines were maintained in high-glucose DMEM medium supplemented with 10 % FBS and 1 % penicillin/streptomycin mixture at 37°C, 5%

Statistical analysis
Data analysis was performed by Excel and GraphPad Prism 6.01 software. For FCM significance analysis, the significance between two means was analyzed using an unpaired two-tailed t-test, and P < 0.05 was considered as significant (**P < 0.01).
Every test was repeated at least for three times.

Availability of data and materials
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.