aKey Laboratory of Polymer Ecomaterials,Changchun Institute of Applied Chemistry,Chinese Academy of Sciences,Changchun 130022,China

bInstitute of Molecular Sciences and Engineering,Shandong University,Qingdao 266237,China

Various calcium ion (Ca2+) nanomodulators are designed for multimodal cancer treatment with the mechanism of intramitochondrial Ca2+overload-induced multilevel mitochondrial destruction.This perspective briefly introduces the development of Ca2+nanomodulators in cancer therapy based on two recent studies published by our research group.

Taking advantage of accurate drug delivery and reduced side effects,subcellular organelle-targeted nanoformulations have attracted more and more attention from cancer therapists.As an essential organelle of mammalian cells,mitochondria play a crucial role in energy conversion,tricarboxylic acid cycle,apoptosis,oxidative stress,calcium ion (Ca2+) storage,and so on.Among these,Ca2+storage is an indispensable work.As one of the key second messengers in the cells,Ca2+participates in a wide range of physiological processes,such as the control of biomembrane permeability and cell excitability,cell metabolism,maintenance of cell morphology,cell cycle regulation,and so froth,and is the hub of a variety of cell signal transmission pathways.Typically,bound calcium and free Ca2+are keeping in a dynamic balance.Once the free Ca2+increases sharply under some conditions,the balance of intramitochondrial Ca2+is broken,leading to cell apoptosis.However,the Ca2+signaling pathway in cancer cells is easily changed by certain drugs,making them more sensitive to the increase of Ca2+concentration than that of normal cells.Hence,intramitochondrial Ca2+overload,which disrupts mitochondrial Ca2+homeostasis,may be an effective strategy for precision cancer therapy[1,2].

We recently developed a multichannel Ca2+nanomodulator (PEGCaNMCUR+CDDP) to boost Ca2+overloadmediated mitochondrial dysfunction in cancer treatment,as shown in Scheme 1A [3].A Ca2+enhancer curcumin (CUR),which increased mitochondrial Ca2+level and inhibited Ca2+efflux,and a mitochondrial dysfunction drug cisplatin(CDDP),which induced mitochondrial damage,were coencapsulated intoPEGCaNMCUR+CDDP.Comparing to other reported a2+nanomodulators,PEGCaNMCUR+CDDP have the following advantages:(a) Simple synthesis steps and high drug loading efficiency,(b) tumor microenvironmenttriggered gradual release behavior with enhanced therapeutic efficacy and reduced systemic toxicity,(c) realized multilevel mitochondrial damage,and(d)excellent fluorescence and PA imaging capacities.

Scheme 1-Various calcium nanomodulators for mitochondria-targeted multimodel cancer therapy.

After intravenous injection,the monodisperse spherical nanoparticlePEGCaNMCUR+CDDPsuccessfully accumulated into the human MCF-7 breast cancer xenograft model through the enhanced permeability and retention (EPR) effect after detachment of poly(ethylene glycol) (PEG) and enhanced cell uptake.A mass of released Ca2+with the assistance of CUR and CDDP achieved multilevel mitochondrial dysfunction.The decreased mitochondrial membrane potential and number of mitochondria,the severest destruction of mitochondrial morphology,the lowest intracellular adenosine triphosphate(ATP) level,and the highest expression of apoptosis proteins were revealed in thePEGCaNMCUR+CDDPgroup,and all the results indicated thatPEGCaNMCUR+CDDPwas an efficient Ca2+nanomodulator for enhanced cancer treatment.

Although Ca2+nanomodulators have been developed for tumor treatment through activating mitochondrial apoptosis pathwaysviamitochondria Ca2+overload,the specific mechanism has not been proven.For the first time,the RNA obtained fromPEGCaNMCUR+CDDP-treated MCF-7 cells was sequenced.The results showed that the positive correlated genes of thePEGCaNMCUR+CDDPgroup were mainly in the terms like “positive regulation of cell death”,“cellular carbohydrate metabolic process”,and“anion transport”,while negative correlated genes were primarily in some biological processes like “mitochondrial gene expression”,“mitochondrial transport”,and “negative regulation of cell cycle”.Therefore,PEGCaNMCUR+CDDPcould activate mitochondrial apoptosis pathwaysviamitochondria Ca2+overload.In addition,this nanoplatform possessed excellent fluorescence and PA imaging capacities.Hence,the multifunctional Ca2+nanomodulatorPEGCaNMCUR+CDDPis a promising organelle-targeted theranostics nanoplatform for cancer treatment.

The successful application of these Ca2+nanomodulators in cancer chemotherapy pushed us to explore whether they can activate antitumor immune or not.Immunogenic cell death(ICD),which can activate antitumor immune responses[4,5],received plenty of focus in cancer immunotherapy [6,7].Hence,in our published study [8],an acid-sensitive PEGdecorated Ca2+nanomodulator(PEGCaCUR)immunogenic cell death (ICD)-inducing properties,as shown in Scheme 1B.Different fromPEGCaNMCUR+CDDP,PEGCaCUR was synthesized without PDA and CDDP.After being combined with ultrasound(US),an exogenous physical stimulus,which could upregulate the intracellular Ca2+concentration,PEGCaCUR+US led to an enhanced Ca nanomodulator.

The monodisperse,spherical,and amorphous nanoparticlesPEGCaCUR released a mass of Ca2+and CUR at intracellular low pH conditions to caused mitochondrial dysfunction through inducing mitochondrial Ca2+overload,featured by lower mitochondrial membrane potential,fewer mitochondria,and more severe destruction of mitochondrial morphology.

For detecting the ICD-inducing properties ofPEGCaCUR,the levels of calreticulin (CRT),high-mobility group box 1(HMGB1),and adenosine triphosphate (ATP) were detected.Interestingly,the cells treated withPEGCaCUR showed CRT exposure and elevated release of HMGB1 and ATP.However,more cell-surface CRT exposure and higher extracellular HMGB1 and ATP levels were found in thePEGCaCUR+US group.All these results verified that mitochondrial Ca2+overload could induce significant ICD,which we could further improve.Then,we proved that reactive oxygen species(ROS)generated by the mitochondrial Ca2+overload contributed to the happen of ICD,and more ROS generation in thePEGCaCUR+US group evoked enhanced ICD efficacy.After six-time treatments,PEGCaCUR exhibited a moderate immune activation effect.As expected,PEGCaCUR+US activated more efficient antitumor immune responses,resulting in effectively suppressing tumor growth and metastasis.

Although these two Ca2+nanomodulators exhibited excellent efficacy of tumor therapy,some issues should be further dissolved in the future.The content of Ca2+inPEGCaNMCUR+CDDPandPEGCaCUR were lower compared with the extracellular fluid.Except for the generation of free Ca2+in the cells and inhibition of efflux,inducing a large influx of extracellular Ca2+is an effective way to cause mitochondrial Ca2+overload.Hence,it’s urgent to develop a new Ca2+nanomodulator with multifunction for more efficient Ca2+accumulation in mitochondria.In addition,expanding the application range of Ca2+nanomodulator is meaningful.

In summary,the developed Ca2+nanomodulators could efficiently inhibit the progression of cancers by inducing significant mitochondrial Ca2+overload with multimodal imaging,which could cause the increased level of intracellular ROS and robust ICD,indicating their great potential for the theranostics of cancers in clinic.

Conflicts of interests

The authors report no conflicts of interest.The authors alone are responsible for the content and writing of this article.

Acknowledgments

This work was financially supported by the National Natural Science Foundation of China (Grant Nos.52022095 and 51873207),the Science and Technology Development Program of Jilin Province (Grant No.20200404182YY),the Youth Innovation Promotion Association of Chinese Academy of Sciences (Grant No.2019230),and the China Postdoctoral Science Foundation(Grant No.2021M691919).