A tumor‐targeting nano‐adjuvant for in situ vaccine based on ultrasound therapy

Ultrasound‐generated antigens combined with TLR7/8 agonists as adjuvants have demonstrated significant anti‐tumor efficacy as an in‐situ vaccine. However, the use of TLR7/8 agonists can cause severe inflammatory responses. In this study, we present a novel tumor‐targeting nano‐adjuvant termed aPDL1‐PLG/R848 NPs, which are composed of aPDL1 antibody, Fc‐III‐4C peptide linker (Fc‐linker) and poly(L‐glutamic acid)‐grafted‐R848. Under ultrasound irradiation, antigen‐presenting cells activate immune mechanisms in vivo under dual stimulation of in situ antigens and immune adjuvants. The strategy inhibits primary tumor growth and induces a strong antigen‐specific immune memory effect to prevent tumor recurrence in vivo. This work offers a safe and potent platform for an in situ cancer vaccine based on ultrasound therapy.


INTRODUCTION
[3] Recently, external physical stimuli such as radiotherapy (RT), [4] photodynamic therapy (PTT), [5] ultrasound therapy [6] have been reported to generate the ISV effect.Ultrasound therapy has attracted much attention as an emerging non-invasive method. [7]The thermal and mechanical effects of ultrasound therapy induce immunogenic cell death (ICD) which in turn releases damage-associated molecular patterns (DAMPs) signals and TAAs, resulting in increased lymphocyte infiltration and activating the antitumor immune response. [8]However, the induction of ICD alone mostly fails to create an effective antitumor response due to insufficient release of antigen or danger signal. [9,10]he development of higher-order combination protocols to ensure additional recruitment and activation of antigenpresenting cells (APCs) as well as the overcoming of the immunosuppressive tumor microenvironment represents the key to success. [11,12]oll-like receptors (TLRs) are the important mediators of inflammatory pathways in cancer immunotherapy. [13]oll-like receptor 7/8 (TLR7/8) is located in the intracellular endosomes, participates in tumor immune surveillance, and plays different roles in tumor growth.When activated, TLR7/8 triggers a Th1-type innate immune response.In addition, they influence T cell function and act as costimulatory molecules, enhancing both innate and adaptive antitumor responses. [14]Resiquimod (R848) is a potent dual TLR7/8 agonist, that has recently gained attention as a cancer immunotherapy agent due to its ability to reprogram immunosuppressive tumor microenvironment. [15]However, its poor solubility and high systemic toxicity (such as skin redness, nausea, fever, headache, shivering, and lymphopenia) hinder its widespread application. [16,17]herefore, to harness its anti-tumor immune effects while minimizing toxicity and enhancing efficacy, researchers have long sought to achieve tumor-selective delivery of R848.
To enhance the local therapeutic efficacy of tumor ultrasound therapy and overcome the bottleneck of the use of R848, we developed a drug delivery system that can selectively deliver R848 to tumors, and a novel tumor-targeting nano-adjuvant termed aPDL1-PLG/R848 NPs was prepared.The aPDL1-PLG/R848 NPs consist of three components: anti-programmed cell death protein 1 antibody (aPDL1 antibody).Fc-III-4C peptide linker (Fc-linker) and poly( Lglutamic acid)-grafted-R848.Due to the high expression of PDL1 in various tumors, the aPDL1 antibody was selected to deliver R848 agonist to tumors, thereby effectively avoiding the systemic inflammatory reactions caused by R848 and achieving a target therapy of tumors.Through the affinity interaction between crystallizable fragment (Fc) of aPDL1 antibody with Fc-III-4C peptide (on the Fc-linker) [18][19][20] and click reaction between azide groups (on the poly( L -glutamic acid)-grafted-R848) and DBCO groups(on the Fc-linker), the aPDL1-PLG/R848 NPs can be easily obtained in an aqueous environment.The method does not affect the antigen-binding fragment (Fab) of an aPDL1 antibody, thus ruling out the possibility of affecting the PDL1 recognization function of the Fab segment. [21]As shown in Scheme 1, the ultrasound-ISV effect generates ICD signals and TAAs first, which with the help of aPDL1-PLG/R848 NPs could induce robust antitumor immune responses.In the MC38 murine colon cancer model, the tumor suppression rate (TSR) and complete response rate (CR) of aPDL1-PLG/R848 NPs (+US) group were 97.3% and 50.0%, respectively, and the tumor-free rate in immune rechallenge was 63.6%.These results indicate that this strategy can effectively activate the anti-tumor immune response and effectively inhibit re-invasion through the anti-tumor immune memory effect.

Verification ISV of ultrasound therapy
To verify whether ultrasound irradiation can produce tumor in situ vaccine effects, in vitro and in vivo experiments were performed.First, the effect of ultrasound treatment on tumor cell viability was verified at the in vitro level.CCK8 assays showed that the ultrasound irradiation time had a significant effect on cell viability.A dose of 3 min ultrasound caused cell viability to decrease below 50%, and a dose of 5 min resulted in cells approaching a non-viable state (Figure S1).DAMPs are molecules released by immunogenic death cells.These DAMPs include but are not limited to, calreticulin (CRT), adenosine 5′-triphosphate (ATP), and high-mobility group 1 (HMGB1).During the initial stage of ICD induction, CRT translocates to the surface of cells as an "eat me" signal.Then, ATP is released or secreted outside the cell, acting as a potent "find me" signal.Similarly, during the later stage of cell death, extracellular release of HMGB1 serves as a "dangerous" signal that stimulates immune response.After ultrasound irradiation, the indicators of DAMPs such as CRT, ATP, and HMGB1 showed different degrees of upregulation (Figure 1B and Figure S9).These released DAMP signals are eventually recognized and activated by APCs to recruit more APCs and T cells. [22]Further, to verify the level of ultrasound-related immunogenicity, its effect on bone marrow dendritic cells (BMDCs) was explored.BMDCs were co-incubated with treated tumor cells.As can be seen from Figure 1C, the activation markers of CD80, CD86, and MHC-II on BMDCs were significantly upregulated after incubation with treated MC38 cells.In vitro experiments have shown that ultrasound-induced ICD effects can activate APCs.APCs recognizing signals from DAMPs or TAAs in vivo are activated to mature and reflux to lymph nodes to stimulate naïve T cells.During the process, APCs upregulate co-stimulatory molecules and produce pro-inflammatory cytokines.Therefore, tumor in situ inflammation is elevated due to increased tumor immunogenicity, activation and maturation of APCs, and invasion of T cells after ultrasound therapy.After treatment in the MC38 model, it was found that the inflammatory factors interleukin (IL)-6 and granzyme B (Gra-B) in situ colon tumors were significantly upregulated.IL-6 is released by activated APCs after activation, and Gra-B secreted by effector T cells plays an irreplaceable role in the process of tumor killing (Figure 1E,F).However, other inflammatory factors such as tumor necrosis factor-alpha (TNF-α), IL-12p70, and interferon-gamma (IFN-γ) were not elevated in tumors.In serum, there were no significant changes in inflammatory factors except for Gra-B.This suggests that ultrasound therapy alone is not enough to induce an effective anti-tumor immune response.
To further demonstrate the effectiveness of ultrasound therapy as an ICD-inducer, a gold-standard method of ICD detection, a vaccine experiment was performed. [23]Prior to the homogenous tumor challenge, mice were vaccinated with nonimmunogenic dead cells (boiled water-treated cells) or MC38 cells that had been treated with ultrasound irradiation on day 0. The results in Figure 1G-I show that vaccination with non-immunogenic dead cells resulted in limited protection against tumor occurrence and growth.Notably, vaccination with ultrasound-treated cells delayed and inhibited the subsequent tumor growth, and exerted a prophylactic effect in 14.3% of mice.Together with studies of in vitro and in vivo expression, our results well demonstrated that ultrasound can be used as an ICD inducer.
Despite its high ability to induce ICD, ultrasound therapy alone struggles to effectively activate systemic anti-tumor immunity.This is primarily due to the fact that the induction of ICD often fails to generate an effective antitumor response due to a lack of sufficient antigen or danger signal release.Therefore, the development of advanced combination protocols that ensure the additional recruitment and activation of APCs, as well as overcoming the immunosuppressive tumor microenvironment, is crucial for successful outcomes.Immunological adjuvants are essential for successful cancer immunotherapy and vaccination.However, traditional adjuvants have some limitations, such as R848 have certain limitations, including a lack of controllability and induction of systemic toxicity, which limits their widespread application.Therefore, the targeted delivery of R848 to the tumor microenvironment has emerged as the preferred option to improve the efficacy of tumor immunotherapy while reducing systemic toxicity.

S C H E M E 1
Schematic diagram illustrates the tumor-targeting nano-adjuvant aPDL1-PLG/R848NPs, which are used for an in situ vaccine based on ultrasound therapy.This form of therapy is capable of inducing the release of immunogenic cell death (ICD) signals and tumor-associated antigens (TAAs) by tumor immunogenic death.After injection, aPDL1-PLG/R848NPs were enriched at the tumor site due to the specific recognition of the aPDL1 antibody.Meanwhile, the immature dendritic cells (DCs) recognized ICD signals and TAAs, and uptake R848, thereby transitioning to an activated phenotype.Mature DC cells then migrate to lymph nodes and stimulated naïve T cells to generate tumor-specific CD8 + T cells.Thus, a strong systemic antitumor immune response was initiated via ultrasound local therapy and aPDL1-PLG/R848 NPs.

Preparation and characterization of aPDL1-PLG/R848 NPs
The tumor-targeting nano-adjuvant is composed of three parts: poly( L -glutamic acid)-grafted-R848 (PLG/R848), Fc-III-4C peptide linker (Fc-linker) and aPDL1 antibody (Figure 2A).[26] In Figure 2B, the 1 H NMR spectrum of PLG/R848 revealed four hydrogen peaks corresponding to the benzene ring of R848, which were observed in the range of δ 7.0 ppm to 8.5 ppm, and the UV wavelength of maximum absorption peak (λ max ) exhibited a red shift (specifically from 320 to 327 nm, Figure S2) after R848 was bonded with PLG chain.These results indicated that the PLG/R848 has been obtained successfully.
The Fc-linker was obtained from the reaction of Fc-III-4C peptide and NHS-PEG 2k -DBCO.As can be seen in the 1 H NMR spectra (Figure S3A), the methylene absorption peaks of NHS disappeared, the molecular weight of Fc-linker was within the range of 3.7 k to 4.0 k (Figure S3B), and the peak at 1100 cm −1 of PEG appeared in the infrared spectrum of Fc-linker (Figure 2C), affirming the effective preparation of Fc-linker.
Then, Fc-linker-PLG/R848 was prepared by the click reaction of azido groups (on the PLG/R848) with DBCO groups (on the Fc-linker), and it can be seen that the DBCO absorption band at 309 nm vanished (Figure 2D).
The 1 H NMR spectrum of Fc-linker-PLG/R848 shows an enhanced characteristic peak of methylene (-CH 2 CH 2 O-) on PEG, along with signals (δ 7.0-8.5 ppm) assigned to hydrogens in the indole ring of Fc-III-4C peptide and the benzene ring of R848 (Figure S4).These illustrated that the Fc-linker-PLG/R848 was successfully prepared.
The aPDL1-PLG/R848 NPs were obtained based on the affinity of aPDL1 and Fc-linker-PLG/R848.The binding of aPDL1 with Fc-linker-PLG/R848 was analyzed using particle size changes.Dynamic light scattering results indicated that the diameters of aPDL1 and Fc-linker-PLG/R848 were 7.5 nm and 91.3 nm, respectively.In contrast, aPDL1-PLG/R848 showed no volume distribution at the diameters of either aPDL1 or Fc-linker-PLG/R848, but almost 100% volume distribution was observed at 142.0 nm (Figure 2E,G).Meanwhile, aPDL1-PLG/R848 NPs exhibited good stability in phosphate-buffered saline (PBS) and serum (Figure S8).The Zeta potential of aPDL1-PLG/R848 NPs was negative (Figure 2F).

In vitro/vivo targeting of MC38 tumor and biodistribution study
[29] Hence, aPDL1 antibodies have been used as effective anti-tumor agents in the treatment of colon cancer tumors. [30]33] Firstly, we investigated the PDL1 expression levels of murine MC38 cancer cell lines and explored the binding effect between aPDL1 antibody and Fc-linker-PLG/R848.[20] The flow cytometry results showed that aPDL1 antibody and Fc-linker-PLG/R848 (modified with Cy5-amine) had high affinity (Figure 3A).Fur-thermore, the targeting ability of aPDL1-PLG/R848/Cy5 NPs to MC38 cells and the affinity of aPDL1 antibody with Fc-Linker-PLG/R848/Cy5 were investigated by confocal laser scanning microscopy (Figure 3B).The results not only suggest that Fc-Linker-PLG/R848/Cy5 attached tightly to aPDL1 antibody but also indicated that the tumortargeting ability of aPDL1 was maintained after its chemical attachment to Fc-Linker-PLG/R848/Cy5.
Next, we also explored the ability of aPDL1-PLG/R848 NPs (NPs modified with Cy5-amine) to target solid tumors in the MC38 mouse model.In vivo, imaging of mice revealed that aPDL1-PLG/R848/Cy5 NPs were enriched at the tumor site in a shorter period of time (Figure 3C,D).Notably, unlike untargeted IgG-PLG/R848/Cy5 NPs which were cleared quickly, aPDL1-PLG/R848/Cy5 NPs continued to accumulate at the tumor sites after 24 h.The aPDL1-PLG/R848/Cy5 group exhibited 1.84-and 2.11-fold higher fluorescence intensity than the IgG-PLG/R848/Cy5 group at 8 and 24 h, respectively.Ultrasound therapy has a destructive effect on tumor blood vessels, which can promote the enrichment of drugs in tumors.Therefore, IgG-PLG/R848/Cy5 NPs (+US) were more enriched (Group 3) than non-ultrasound-treated groups (Group 1), with a 1.93-fold difference at 1 h.Consistent with the results above, aPDL1-PLG/R848/Cy5 NPs (+US) were effectively enriched in the tumors at different time points compared to other groups, with approximately 1.58-fold higher enrichment (Group 4) than non-ultrasound treated groups (Group 2) after 12 h of injection.To further investigate the biodistribution of aPDL1-PLG/R848/Cy5 NPs, MC38-bearing mice were euthanized at 24 h postinjection, and major organs were collected subsequently for the ex vivo imaging to quantitatively assess the accumulation of aPDL1-PLG/R848/Cy5 NPs in tissues.All NPs groups were enriched to varying degrees in the heart, liver, spleen, lung, kidney, and lymph nodes, with no significant differences between the groups (Figure 3E,F).Taken together, aPDL1-PLG/R848/Cy5 NPs have good tumor-homing ability and great potential for targeting cancer therapy, while ultrasound therapy will effectively improve its efficiency.

Anti-tumor effect of aPDL1-PLG/R848 NPs with ultrasound therapy in MC38 tumor model
Ultrasound-mediated in situ vaccines for tumors are not sufficient to initiate a robust systemic anti-tumor immune response.Therefore, we hoped to achieve an enhanced ultrasound-mediated ISV by targeted delivery of nanoagonists to the tumor.We selected the MC38 colon tumor model for validation.In our experiments, 2 × 10 6 MC38 cells were inoculated on the left flank of each mouse, and the average tumor size was allowed to reach approximately 100 mm 3 .The mice were randomly separated into six groups (n = 6) and treated with PBS (control, Group 1), PBS (+US) (Group 2), Mix of aPDL1 antibody and PLG/R848 (Mix, Group 3), Mix (+US) (Group 4), aPDL1-PLG/R848 (Group 5), and aPDL1-PLG/R848 (+US) (Group 6), respectively.As shown in Figure 4A, Mice were treated every three days for a total of three treatments, and ultrasound irradiation was performed only single treatment.The body weight and tumor growth of mice were continuously monitored.
The PBS group showed the fastest tumor growth, and the tumor volume reached approximately 1700 mm 3 on day 18 (Group 1 of Figure 4B,D).After ultrasound treatment, the ultrasound therapy group experienced a slower tumor growth than that of the PBS group.This indicated that ultrasound irradiation-induced tumor immunogenic death could activate the immune response in vivo and further inhibit tumor growth by ISV effect.However, its growth rate was only slow and did not fundamentally improve, and its tumor suppression rate was only 33.8% on day 18 (Group 2 of Figure 4B,D).Therefore, it is necessary to provide more effective treatment to activate tumor immunity.To test the proposed scenario, we injected mice with aPDL1 antibody and PLG/R848 after ultrasound irradiation, which were two components of the aPDL1-PLG/R848 NPs.The therapeutic effect of the ISV can also be improved, and its tumor suppression rate was 58.5% (Group 4 of Figure 4B,D).Although the regimen of aPDL1 antibody did not provide R848 immune adjuvant for tumor enrichment, it selectively repaired defects in overregulated tumor-specific effector T cells and restored immune competence against tumors by anti-PD-1/PD-L1 antibodies to block the PD-1/PD-L1 signaling pathway.PLG/R848 can also activate DCs to enhance tumor-specific effector T cells mediated anti-tumor immune response.Consistent with the proposed scenario, the injection of aPDL1-PLG/R848 NPs after ultrasound therapy could effectively inhibit the tumor growth, with a tumor suppression rate and cure rate of 97.3% and 50%, respectively (Group 6 of Figure 4B,D).The aPDL1 antibody not only blocks the PD immune pathway but also enables R848 to be delivered to the tumor site with immunogenicity after ultrasound treatment.Therefore, DCs in the tumor microenvironment can phagocytose or recognize tumor cells, and activate DCs to enhance tumor-specific effector T cells mediated anti-tumor immune response.Up to the end of 60 days, the tumor-free mice had no tumor recurrence and the others had no tumor growth (Group 6 of Figure 4C).No significant body weight or liver and kidney function change occurred during the treatment (Figures S5 and S10).The results of tumor hematoxylin and eosin staining and immuno-fluorescence sections were as expected.
From Figure 4E, it can be seen that the tumors in group 6 have large necrotic areas, and the degree of tumor invasion of CD3 + CD8 + T cells is greatly increased.These results once again illustrate that ultrasound-mediated tumor in situ vaccine combined with aPDL1-PLG/R848 NPs can effectively activate the anti-tumor immune response and effectively inhibit the growth of tumors.

Long-term immune memory effect against rechallenged pathogen
Based on the excellent antitumor efficacy of aPDL1-PLG/R848 NPs with ultrasound local therapy, it is necessary to evaluate whether this treatment approach can induce tumor-specific memory responses in the body and initiate systemic antitumor effects.Therefore, we explored the rechallenge of immunization to investigate the immune memory effect induced by ultrasound therapy with aPDL1-PLG/R848 NPs.
[36] In our study, mice without detectable tumors after surgery or treatment were rechallenged with the second batch of cancer cells 30 days later.The results showed that the combination of ultrasound-mediated in situ vaccine and aPDL1-PLG/R848 NPs indeed produced a tumor-specific immune memory effect.When tumor cells carrying the same antigen infiltrated, they elicited robust and specific immune responses.This synergy between ultrasound-mediated ISV and aPDL1-PLG/R848 NPs effectively promoted tumorspecific immune memory.
To explore the immune memory effect induced by ultrasound therapy with aPDL1-PLG/R848 NPs, we rechallenged mice without detectable tumors after surgery or treatment 30 days later with a second batch of cancer cells.Mice were divided into two groups (n = 11): (1) surgery (control, Group 1) and (2) aPDL1-PLG/R848 NPs (+US) (Group 6).In Group 6, aPDL1-PLG/R848 NPs (consisting of 100 μg of aPDL1 antibody and 6.8 μg of R848 per mouse) were injected four times (on days 8, 11, 14, and 17) along with a single treatment of ultrasound (Figure 5A).Then, MC38 cells were inoculated on day 30 and continuously observed for tumor formation or growth.By day 50, the average tumor volume in Group 1 reached 626.9 mm 3 , whereas only four mice in Group 6 had solid tumors with an average volume of 173.8 mm 3 (Figure 5B).No tumors were generated in the remaining seven mice in Group 6.These findings underscore the effective generation of tumor-specific immune memory through the combined approach of ultrasound-mediated ISV and aPDL1-PLG/R848 NPs.Furthermore, when tumor cells carrying the same antigen infiltrated, they elicited robust and specific immune responses.
To investigate the mechanism of antitumor immune memory induced by the treatment, we analyzed tumor-draining lymph nodes (TDLNs), spleens of immune cells, and serum cytokines on day 35 in two groups of mice.When the antigen re-exposures, antigen-presenting DC cells immediately recognize the antigen and migrate back into the lymph nodes, thereby stimulating T cells to initiate a specific tumor immune response.In the TDLN of Group 6 mice, the CD11c + CD80 + cells (activated DC cells) and CD3 + CD8 + T cells were upregulated to varying degrees (Figure 5F,G).In the spleens of Group 6 mice, the types of central memory T cells (T CM , characterized by CD3 + CD8 + CD62L + CD44 + ) and effector memory T cells (T EM , characterized by CD3 + CD8 + CD62L − CD44 + ) exhibited greater expression (Figure 5C-E).These memory T cell subsets expanded and produced important cytokines at the beginning of antigen stimulation and were more expressive.Likewise, much higher levels of serum cytokines such as IFN-γ, TNF-α, IL-12p70, and IL-6, which play vital roles in cellular immunity against cancer, were observed (Figure 5H).Thus, the treatment indeed has the potential to induce strong immune memory that can effectively inhibit the recurrence of cancer.

CONCLUSION
In conclusion, the study developed a tumor-targeting nanoadjuvant called aPDL1-PLG/R848 NPs for ultrasound therapy.The nano-adjuvant was tested on MC38 tumors, achieving a tumor suppression rate of 97.3% and a complete response of 50.0%.The survival rate was 100% on day 60 without any obvious side effects.The nano-adjuvant also allowed the body to generate a potent immune memory effect to resist the same stress.This study suggests that the strategy of delivering R848 with aPDL1 antibody could be potentially used in an in situ vaccine by ultrasound therapy.

A C K N O W L E D G M E N T S
This work was supported by the Ministry of Science and Technology of China (Project 2022YFE0110200), the Natural Science Foundation of Jilin Province (Project 20230101037JC), and the National Natural Science Foundation of China (Projects 52203198 and 52025035).

F I G U R E 1
In vitro and in vivo verification of ultrasound immunogenicity.(A) Schematic illustration of testing immunogenicity after ultrasound irradiation in vitro.(B) Detection of immunogenic cell death (ICD) (calreticulin [CRT], adenosine 5′-triphosphate [ATP], and high-mobility group 1 [HMGB1]) markers after ultrasound irradiation.(C) Activation of BMDCs after ultrasound irradiation.(D) Schematic illustration of serum and solid tumor testing for pro-inflammatory cytokines after ultrasound irradiation in vivo.(E, F) Pro-inflammatory cytokines of tumor and blood serum after ultrasound irradiation.(G-I) In vivo prophylactic vaccination experiments.MC38 cells were treated by ultrasound for 24 h and then injected s.c.into one flank of C57BL/6 mice (n = 7 per group).On day 3, mice were challenged with living MC38 cells at the contralateral flank.Tumor incidence and growth were monitored.Data are represented as mean ± SEM.Statistical significance was determined by Student's t-test.(ns: no significance, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

F I G U R E 4
Anti-tumor effect of aPDL1-PLG/R848 NPs for in situ vaccine based on ultrasound therapy.(A) Schematic illustration of the experimental design to assess the in vivo.A common legend is presented at the top left of Figure 4. (B) Tumor growth curves, (C) survival curves, and (D) detailed tumor suppression curves of each mouse (n = 6 per group).(E) Hematoxylin and eosin (H&E) and IF of tumors from different groups.Data are represented as mean ± SEM.Statistical significance was determined by a one-way analysis of variance (ANOVA) test.(ns: no significance, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

F I G U R E 5
Immune rechallenge of aPDL1-PLG/R848 NPs with ultrasound therapy in MC38 tumor model.(A) Schematic illustration of the experimental design to assess the in vivo.A common legend is presented at the top left of Figure 5. (B) Tumor growth curves of different groups (n = 11 per group).(C-E) Proportions of effector memory T cells (T EM ) and central memory T cells (T CM ) in the spleen analyzed by flow cytometry (gated on CD3 + CD4 + T cells and CD3 + CD8 + T cells) at day 35 (n = 3 per group).(F, G) Proportions of CD3 + CD8 + T cells and CD11c + CD80 + cells in the TDLN at day 35.(H) Cytokine levels in serum from different groups of mice at day 35 (n = 3 per group).Data are represented as mean ± SEM.Statistical significance was determined by Student's t-test.(ns: no significance, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).