Preparation Of Targeted Nanomedicines Capable Of Breaking The Tumor Dense Matrix Barrier And Evaluation Of Their Efficacy

BackgroundMalignant tumors are a serious threat to human health and life.Chemotherapy is one of the main treatments for them.Intra-tumor drug delivery barriers severely reduce the efficiency of tumor stromal transport and vascular transport of drugs,thus preventing efficient penetration of drugs into tumor cells,resulting in low drug delivery efficiency,which cannot effectively kill tumor cells,but easily cause tumor recurrence,progression and drug resistance.Tumor drug delivery barriers include tumor pathological vasculature and dense extracellular matrix in tumor.A variety of therapeutic interventions have emerged to address different tumor drug delivery barriers,including vascular normalization strategies targeting tumor pathological vessels,synthesis inhibition and catabolism promotion strategies targeting major components of the tumor extracellular matrix（e.g.hyaluronic acid,HA）,and some mechanical fragmentation and thermal therapies to disrupt the tumor dense matrix barrier.Although different therapeutic strategies have achieved certain research results in animal or clinical trials,they still reveal the problem of lack of targeting,leading to some toxic side effects that limit the clinical application of some drugs and other problems.Therefore,how to better solve the problem of intra-tumor drug delivery barrier to enhance drug delivery efficiency and reduce toxic side effects is still a huge problem for clinicians to solve in anti-tumor therapy,and a lot of basic research and clinical applications are still needed to be explored and tested.It is hoped that through these continuous efforts and new improvements and explorations,the problem of tumor drug delivery barriers can be solved eventually,so that better antitumor therapeutic effects can be achieved with lower drug doses and less toxic side effects,which will eventually lead to significant improvements in patient survival and quality of life.Objectives1.The objective of this study was to synthesize a novel highly efficient targeted nanochemotherapeutic drug c（RGDyK）-HAase-IONP/DOX,which targets tumors and degrades a large amount of hyaluronic acid in their dense stroma to dismantle the tumor dense stromal barrier and fundamentally improve the drug delivery efficiency to achieve better tumor suppression.A series of characterizations were performed and the water solubility,stability and enzymatic activity of the novel chemotherapeutic nanodrugs were evaluated,and the drug loading capacity and encapsulation rate were calculated.2.To assess the efficiency of in vitro drug release comparing c（RGDyK）-HAase-IONP/DOX under physiological and acidic p H conditions.3.To evaluate the tumor cell targeting of c（RGDyK）-HAase-IONP nanocarriers and to assess the cytotoxic effects of c（RGDyK）-HAase-IONP/DOX nanodrugs and corresponding nanocarriers.4.To investigate the in vivo targeting,biodistribution and intra-tumor delivery of c（RGDyK）-HAase-IONP,and to evaluate the anti-tumor therapeutic effect and in vivo biosafety of this novel highly effective targeted nano-chemotherapy drug.Methods1.The highly efficient targeting nano-drug carrier c（RGDyK）-HAase-IONP was firstly constructed,and then the hydrophobic chemotherapeutic drug DOX was loaded into the hydrophobic space of the amphiphilic polymer coating of IONP by physical embedding to prepare the highly efficient tumor-targeting nano-chemotherapeutic drug c（RGDyK）-HAase-IONP/DOX capable of dismantling the tumor dense stromal barrier.The morphology,particle size,potential and absorption and emission spectra were characterized by applying TEM,DLS,potential analyzer and fluorescence spectrophotometer.The enzymatic activity of c（RGDyK）-HAase-IONP/DOX was verified by turbidimetric method.The drug loading rate and encapsulation efficiency of DOX were determined by HPLC.In addition,we dissolved it into saline,water,PBS and DMEM medium containing 10%FBS,respectively,and stored it at room temperature at rest.After 7 days,the samples were digitally photographed and the nanodrug hydrodynamic size and particle size distribution were determined by DLS to explore its solubility and stability in different physiological dispersion media.2.Thepotenttumor-targetingnanochemotherapeuticdrug c（RGDyK）-HAase-IONP/DOX was dissolved in PBS solutions with p H 7.4 and 5.5,respectively,and transferred to dialysis bags.The dialysis bags were then immersed in 50m L centrifuge tubes containing 20 m L of p H 7.4 and 5.5 PBS solutions,respectively,and shaken lightly at 110 rpm with a 37°C thermostatic shaker.At each pre-set time point,0.2m L of the solution was removed from the 20 m L PBS solution to be measured and replenished with an equal amount of the corresponding PBS.The cumulative release of DOX at each time point was measured and calculated by HPLC and analyzed to investigate the changes in drug release efficiency in both groups to assess the in vitro drug release efficiency of c（RGDyK）-HAase-IONP/DOX under physiological and acidic p H conditions.As a control,drug release from DOX alone and IONP/DOX was also studied using the same methods described above.3.To investigate the targeting of the novel nanocarrier c（RGDyK）-HAase-IONP,we selected murine-derived colon cancer cells MC38 expressing integrinα_vβ₃ and incubated them with the NIR fluorescent dye ICG-labeled c（RGDyK）-HAase-IONP and ICG-labeled IONP,respectively.To further validate the targeting of c（RGDyK）to integrinαvβ3 on the tumor cell surface,we set up a c（RGDyK）blocking group.Targeted binding of each group of cells was observed by confocal microscopy.In the study of cytotoxicity experiments,we used murine-derived colon cancer cells MC38 in logarithmic growth phase and in good growth condition,and the toxicity experiments were divided into four groups:DOX-only group.IONP/DOX group,HAase-IONP/DOX group and c（RGDyK）-HAase-IONP/DOX group.We performed toxicity studies with drug（DOX）concentrations of 0.1,10,100,200,300 and 900 ng/m L,respectively.After different treatments and incubation by each group,100μL of medium solution containing 10%Alamar Blue was added and incubated for about3 h.The absorbance values at 590 nm were measured by enzyme marker.The cell survival rate under the effect of different concentrations of drug was also calculated.In addition,the cytotoxicity assay of each nanocarrier against MC38 was also studied by the same method.4.MC38 tumor-bearing mouse model was constructed.First,we stained MC38 tumor tissues with HA antibodies to indicate the presence of large amounts of HA in the tumor ECM,which provided an experimental basis for the introduction of HAase intervention strategy.Then,to further determine the effect of different nanocarriers in the tumor and their specific distribution in the tumor,the tumor-bearing mice were randomly divided into three groups:ICG-IONP group,ICG-HAase-IONP and ICG-c（RGDyK）-HAase-IONP group,and the corresponding nanocarriers（the amount of IONP was 400 pmole）were injected via tail vein for 100μL of ICG-labeled nanocarriers.In vivo fluorescence signal detection was performed by a small animal in vivo imaging system under isoflurane anesthesia 24 h after tail vein injection to compare the tumor targeting,intratumoral accumulation and biodistribution of the three groups of nanocarriers in tumor-bearing mice.In the intratumoral delivery effect and localization analysis study,we performed HE staining of frozen tumor tissue sections to observe the tumor histomorphology and immunofluorescence staining of relevant molecular markers（CD31,CK19）to facilitate more visual analysis of the relationship between nanocarriers and the location of blood vessels and tumor cells,and used fluorescence confocal microscopy with NIR fluorescence signal confocalization to localize intra-tumor distribution of different ICG-labeled nanocarriers.We also used MC38tumor-bearing mouse model for the evaluation of antitumor treatment efficacy.The tumor-bearing mice were randomly divided into five groups:（1）DOX-only group,（2）IONP/DOX group,（3）HAase-IONP/DOX group,（4）c（RGDyK）-HAase-IONP/DOX group,and（5）saline control group,with five mice in each group.The tumor volumes and body weights of the mice were monitored every other day,and the mice were executed on day 16after the initial treatment.The tumors and major organs of each group were stained with HE,immunofluorescence staining with Ki67,cleaved caspase-3 and HA antibodies,and analyzed to assess the anti-tumor treatment effects and in vivo biosafety of each group.Results1.The targeted nanocarrier c（RGDyK）-HAase-IONP,which can dismantle the tumor dense matrix barrier,was successfully constructed and loaded with hydrophobic chemotherapeutic drug DOX to produce highly efficient targeted nanochemical drug c（RGDyK）-HAase-IONP/DOX.Thehydratedparticlesizeof c（RGDyK）-HAase-IONP/DOX was about 27 nm with a relatively homogeneous particle size and a zeta potential of about 6.5 m V,and it demonstrated good stability in different physiological solutions with a drug loading of 21.7%.We also demonstrated the enzymatic activity of c（RGDyK）-HAase-IONP/DOX by turbidimetric method.2.In in vitro drug release experiments,by control we found that c（RGDyK）-HAase-IONP/DOX was effective in slowing drug release compared to DOX alone and demonstrated different drug release efficacy under physiological and acidic p H conditions.Under acidic conditions at p H 5.5,the drug release rate was faster and the cumulative drug release was higher,which would facilitate rapid and efficient drug release in the acidic tumor microenvironment.3.Targeted uptake experiments showed that the novel highly efficient targeting nanocarrier c（RGDyK）-HAase-IONP has good active targeting effect on MC38 tumor cells,thus increasing the targeted delivery of drugs to tumor cells to enhance tumor suppression efficacy and reduce toxic side effects.In the in vitro cytotoxicity assay,we demonstrated that the nanocarrier doses applied in this study have no toxic effects on cells and have good biosafety;DOX encapsulated in the novel highly efficient targeting nanocarrier c（RGDyK）-HAase-IONP did not affect its cytotoxic effect,and c（RGDyK）-HAase-IONP/DOX had good growth inhibition on tumor cells.The c（RGDyK）-HAase-IONP/DOX has a good inhibitory effect on tumor cell growth and has good prospects for in vivo antitumor therapeutic applications.4.Immunofluorescence staining of MC38 tumor tissue sections with hyaluronan antibody confirmed the presence of large amounts of hyaluronan in the tumor extracellular matrix,which would impede efficient delivery of antitumor drugs to tumor cells,and the results of this experiment also provided support for the experimental basis for the strategy of introducing HAase to dismantle the tumor-dense barrier effect in this study.In the in vivo fluorescence imaging,c（RGDyK）-HAase-IONP demonstrated good tumor targeting and could accumulate more at tumor sites,and by co-localization with vascular staining,it was determined that c（RGDyK）-HAase-IONP not only accumulated highly in tumors,but also could effectively penetrate to deeper locations away from blood vessels,which could deliver drugs to more of tumor cells,facilitating full drug efficacy to improve antitumor therapeutic effects.In the in vivo efficacy evaluation,we demonstrated that c（RGDyK）-HAase-IONP/DOX treatment group significantly inhibited tumor growth,had the best anti-tumor therapeutic effect,and effectively degraded dense HA in tumor with good in vivo safety by histological experiments.ConclusionsIn this study,we enhanced the tumor-targeting property of IONP by c（RGDyK）modification and introduced HAase to degrade a large amount of HA barrier in ECM,and successfully constructed a novel highly efficient targeting nanocarrier c（RGDyK）-HAase-IONP loaded with hydrophobic chemotherapeutic drug DOX to obtain a targeting nano-drug c（RGDyK）-HAase-IONP/DOX capable of dismantling the tumor dense matrix barrier.We demonstrated the successful construction of c（RGDyK）-HAase-IONP/DOX through a series of characterization experiments,and demonstrated good water solubility and stability.The targeting,intratumoral delivery,antitumor therapeutic effect and in vivo biosafety were also investigated by in vitro,cellular and animal experiments.The novel highly effective targeted nanomedicine has good tumor targeting properties,which can effectively degrade the dense HA in the tumor,break its drug delivery barrier,and enhance the drug delivery effect in the tumor site,especially in the deep part of the tumor away from blood vessels,thus fundamentally improving the anti-tumor efficacy.