Oncolytic mineralized bacteria as potent locally administered immunotherapeutics.

Oncolytic bacteria can trigger innate immune activity. However, the antitumour efficacy of inactivated bacteria is poor, and attenuated live bacteria pose substantial safety risks. Here we show that intratumourally injected paraformaldehyde-fixed bacteria coated with manganese dioxide potently activate innate immune activity, modulate the immunosuppressive tumour microenvironment and trigger tumour-specific immune responses and abscopal antitumour responses. A single intratumoural administration of mineralized Salmonella typhimurium suppressed the growth of multiple types of subcutaneous and orthotopic tumours in mice, rabbits and tree shrews and protected the cured animals against tumour rechallenge. We also show that mineralized bacteria can be administered via arterial embolization to treat orthotopic liver cancer in rabbits. Our findings support the further translational testing of oncolytic mineralized bacteria as potent and safe antitumour immunotherapeutics.

CaCO3 -Encapuslated Microspheres for Enhanced Transhepatic Arterial Embolization Treatment of Hepatocellular Carcinoma.

Transcatheter arterial embolization (TAE) is an extensively applied treatment method for hepatocellular carcinoma (HCC). However, the worsened tumor microenvironment (TME, e.g., reduced pH post-TAE) may result in unsatisfactory therapeutic outcome. Herein, a new kind of embolic agent, calcium carbonate encapsulated alginate microspheres (CaCO3 -ALG MSs) are synthesized. Such CaCO3 -ALG MSs are able to neutralize the tumor pH owing to the reaction of CaCO3 with protons, which would not affect the overall morphology of microspheres after decomposition of CaCO3 . TAE treatment with CaCO3 -ALG MSs is then conducted in an orthotopic rat liver cancer model. 18 F-Fluorodeoxyglucose micropositron emission tomography/computed tomography imaging is conducted post-TAE and discovered that intra-arterial injection of CaCO3 -ALG MSs shows obvious enhanced therapeutic outcome compared to the same treatment with bare ALG MSs or the clinically used lipiodol. Further studies including analysis of immune cells in tumors, cytokine assays, and bioinformatics analysis all verify the reverse of immunosuppressive TME toward a more immunosupportive one after TAE with CaCO3 -ALG MSs. The research not only presents a new CaCO3 -containing embolic agent for enhanced TAE treatment of HCC but also highlights a clinically meaningful approach to improve cancer treatment via tumor pH neutralization.

ATP-Responsive Smart Hydrogel Releasing Immune Adjuvant Synchronized with Repeated Chemotherapy or Radiotherapy to Boost Antitumor Immunity.

Certain chemotherapeutics and forms of ionizing radiation can induce immunogenic cell death (ICD). If there simultaneously exist immune adjuvants within the tumor, such antitumor immunity would be further amplified. However, as clinical chemo/radiotherapies are usually repeatedly given at low individual doses, it would be impractical to administrate immune adjuvants into tumors at each dose of chemo/radiotherapies. Thus, a smart hydrogel is developed that releases immune adjuvants in response to repeatedly applied chemo-/radiotherapies. Herein, alginate is conjugated with an adenosine triphosphate (ATP)-specific aptamer, which is hybridized with immunoadjuvant CpG oligonucleotide. Upon intratumoral injection, alginate-based hydrogel is formed in situ. Interestingly, low doses of oxaliplatin or X-rays, while inducing ICD of tumor cells, could trigger release of ATP, which competitively binds with ATP-specific aptamer to trigger CpG release. Therefore, the smart hydrogel could release the immune adjuvant synchronized with low-dose repeated chemo/radiotherapies, achieving remarkable synergistic responses in eliminating established tumors, as well as immune memory to reject re-challenged tumors. Moreover, repeated radiotherapies assisted by the smart hydrogel could inhibit distant tumor metastases, especially in combination with immune checkpoint blockade. The study presents a conceptually new strategy to boost cancer immunotherapy coherent with repeated low-dose chemo-/radiotherapies following a clinically relevant manner.

Surfactant-stripped J-aggregates of azaBODIPY derivatives: All-in-one phototheranostics in the second near infrared window.

Development of multifacted phototheranostics with bright fluorescence and absorbance in the second near infrared (NIR-II) window is very appealing for precise cancer diagnosis and treatment, but still challenging nowadays. Herein, we synthesize a hydrophobic annularly fused azaBODIPY (termed as HBP) molecule with sharp NIR absorbance peaked at 878 nm and bright NIR-II fluorescence. With Pluronic F127 as the surfactant and hydrophobic paclitaxel (PTX) as the spacer, such HBP molecule would self-assemble to form surfactant-stripped HBP/PTX micelles with absorption peak red-shifted to 1012 nm and intrinsic NIR-II fluorescence negligibly disturbed. We found that such HBP/PTX micelles can be utilized as a bimodal NIR-II nano-probe to enable real-time tracking of lymph nodes and tumors under an NIR-II fluorescence imaging system, as well as clear visualization of tumor microvasculatures under an NIR-II photoacoustic imaging system. Furthermore, together with 1064 nm laser exposure, such HBP/PTX micelles would synergistically suppress the growth of tumors grown on the mice upon tumor accumulation. This work highlights the concise preparation of a type of all-in-one NIR-II phototheranostics from the newly synthesized HBP molecules, thereby enables NIR-II fluorescence/photoacoustic bimodal imaging guided synergistic cancer treatment via the NIR-II laser boosted photothermal therapy and chemotherapy.

Perfluorocarbon nanodroplets stabilized with cisplatin-prodrug-constructed lipids enable efficient tumor oxygenation and chemo-radiotherapy of cancer.

Concurrent chemo-radiotherapy has been widely applied for the treatment of a wide range of cancers, but its therapeutic efficacy against most solid tumors is severely impaired by their intrinsic hypoxic microenvironments. Utilizing the high oxygen loading capacity of perfluoro-15-crown-5-ether (PFCE), herein, we prepare PFCE nanodroplets with cisplatin prodrug (cisPt(iv)) conjugated phospholipids and other commercial lipids as the stabilizer to enable tumor targeted oxygen shuttling. The obtained PFCE@cisPt(iv)-Lip shows high physiological stability and efficient oxygen loading capacity. As vividly visualized under an in vivo photoacoustic imaging system, tumors on the mice with intravenous injection of such PFCE@cisPt(iv)-Lip show effective tumor oxygenation. Together with X-ray exposure, such PFCE@cisPt(iv)-Lip upon intravenous injection could induce severe DNA damage of cells, thereby remarkably suppressing the tumor growth and significantly prolonging their survival time without causing obvious toxic side effects. This work highlights PFCE@cisPt(iv)-Lip as an adjuvant nanomedicine for enhanced chemo-radiotherapy of tumors by attenuating hostile tumor hypoxia, indicating its promising potential for future clinical translation ascribed to its straightforward synthesis and notable tumor growth inhibition at a safe dose.

Localized cocktail chemoimmunotherapy after in situ gelation to trigger robust systemic antitumor immune responses.

Currently, there is a huge demand to develop chemoimmunotherapy with reduced systemic toxicity and potent efficacy to combat late-stage cancers with spreading metastases. Here, we report several "cocktail" therapeutic formulations by mixing immunogenic cell death (ICD)-inducing chemotherapeutics and immune adjuvants together with alginate (ALG) for localized chemoimmunotherapy. Immune checkpoint blockade (ICB) antibody may be either included into this cocktail for local injection or used via conventional intravenous injection. After injection of such cocktail into a solid tumor, in-situ gelation of ALG would lead to local retention and sustained release of therapeutics to reduce systemic toxicity. The chemotherapy-induced ICD with the help of immune adjuvant would trigger tumor-specific immune responses, which are further amplified by ICB to elicit potent systemic antitumor immune responses in destructing local tumors, eliminating metastases and inhibiting cancer recurrence. Our strategy of combining clinically used agents for tumor-localized cocktail chemoimmunotherapy possesses great potential for clinical translation.

BCL11A expression in acute phase chronic myeloid leukemia.

Chronic myeloid leukemia (CML) has chronic and acute phases. In chronic phase myeloid differentiation is preserved whereas in acute phase myeloid differentiation is blocked. Acute phase CML resembles acute myeloid leukemia (AML). Chronic phase CML is caused by BCR-ABL1. What additional mutation(s) cause transition to acute phase is unknown and may differ in different persons with CML. BCL11A encodes a transcription factor and is aberrantly-expressed in several haematological and solid neoplasms. We analyzed BCL11A mRNA levels in subjects with chronic and acute phase CML. BCL11A transcript levels were increased in subjects with CML in acute phase compared with those in normals and in subjects in chronic phase including some subjects studied in both phases. BCL11A mRNA levels were correlated with percent bone marrow blasts and significantly higher in lymphoid versus myeloid blast crisis. Differentiation of K562 with butyric acid, a CML cell line, decreased BCL11A mRNA levels. Cytology and flow cytometry analyses showed that ectopic expression of BCL11A in K562 cells blocked differentiation. These data suggest BCL11A may operate in transformation of CML from chronic to acute phase in some persons.

BCL11A expression in acute myeloid leukemia.

BACKGROUND: BCL11A encodes a C2H2 type zinc-finger protein. During normal haematopoietic cell differentiation BCL11A expression is down-regulated. Data in mice suggest up-regulation of BCL11A is involved in the pathogenesis of myeloid leukaemias. BCL11A expression in persons with acute myeloid leukaemia (AML) is not systematically studied. OBJECTIVE: Interrogate associations between BCL11A expression at diagnosis and clinical and laboratory valuables and outcomes in newly-diagnosed persons with AML. METHODS: We determined BCL11A mRNA levels in bone marrow and blood mononuclear cells in 292 consecutive newly-diagnosed subjects with AML by reverse transcript and real-time polymerase chain reaction. Data were compared to mRNA levels in bone marrow cells of normals. RESULTS: Subjects with BCL11A transcript levels at diagnosis exceeding the median value of 2.434 (±3.423 SD; 25th-75th inter-quartile range, 1.33-4.29) had higher WBC levels, a greater proportion of bone marrow myeloblasts, were more likely to be FAB M0 subtype, less likely to be FAB M3 subtype, more likely to be in the intermediate cytogenetic risk cohort, less likely to have a complex karyotype and more likely to have DNMT3A(R882) and FLT3-ITD mutations than subjects with transcript levels below the median value. In 89 subjects receiving conventional induction chemotherapy the complete remission rate was 54% (95% confidence interval [CI]; 33, 75%) in the lower BCL11A cohort and 65% (45, 85%; P=0.26) in the higher BCL11A cohort. 3 year survival was 33% (2, 65%) in the lower BCL11A cohort and 15% (0, 39%; P=0.35) in the high BCL11A cohort. CONCLUSION: BCL11A transcript levels at diagnosis was significantly associated with several clinical and laboratory variables. There were also non-significant associations with complete remission rate and survival. These data suggest a possible role for BCL11A expression in AML biology.

Aberrant expression of ecotropic viral integration site-1 in acute myeloid leukemia and acute lymphoblastic leukemia.

Ecotropic viral integration site-1 (EVI1) proto-oncogene expression in patients with acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) requires further investigation. Here, EVI1 expression levels were measured in 216 Chinese patients with AML and 67 with ALL via quantitative real-time polymerase chain reaction. We found that EVI1 expressed at a high level (H-EVI1) was present in 11.1% of patients with AML versus 20.9% with ALL. Low levels of EVI1 expression occurred in 23.1% with AML versus 43.3% with ALL. This suggested that alteration of EVI1 expression was more profound in ALL than in AML. H-EVI1 was significantly enriched in 30-60-year-old patients. French-American-British (FAB) M3 subtype was significantly correlated with H-EVI1. Interestingly, we found that EVI1 expression was negatively associated with presence of the Philadelphia chromosome (Ph+) and MLL rearrangements in AML. However, Ph+, but not MLL rearrangements, was inversely correlated with EVI1 expression in B-ALL. These results for the first time suggest a mutually exclusive relationship between EVI1 expression and Ph+ karyotype.

Functionalized graphene oxide in enzyme engineering: a selective modulator for enzyme activity and thermostability.

The understanding of interactions between nanomaterials and biomolecules is of fundamental importance to the area of nanobiotechnology. Graphene and its derivative, graphene oxide (GO), are two-dimensional (2-D) nanomaterials with interesting physical and chemical properties and have been widely explored in various directions of biomedicine in recent years. However, how functionalized GO interacts with bioactive proteins such as enzymes and its potential in enzyme engineering have been rarely explored. In this study, we carefully investigated the interactions between serine proteases and GO functionalized with different amine-terminated polyethylene glycol (PEG). Three well-characterized serine proteases (trypsin, chymotrypsin, and proteinase K) with important biomedical and industrial applications were analyzed. It is found that these PEGylated GOs could selectively improve trypsin activity and thermostability (60-70% retained activity at 80 °C), while exhibiting barely any effect on chymotrypsin or proteinase K. Detailed investigation illustrates that the PEGylated GO-induced acceleration is substrate-dependent, affecting only phosphorylated protein substrates, and that at least up to 43-fold increase could be achieved depending on the substrate concentration. This unique phenomenon, interestingly, is found to be attributed to both the terminal amino groups on polymer coatings and the 2-D structure of GO. Moreover, an enzyme-based bioassay system is further demonstrated utilizing our GO-based enzyme modulator in a proof-of-concept experiment. To our best knowledge, this work is the first success of using functionalized GO as an efficient enzyme positive modulator with great selectivity, exhibiting a novel potential of GO, when appropriately functionalized, in enzyme engineering as well as enzyme-based biosensing and detection.

In vivo NIR fluorescence imaging, biodistribution, and toxicology of photoluminescent carbon dots produced from carbon nanotubes and graphite.

Oxidization of carbon nanotubes by a mixed acid has been utilized as a standard method to functionalize carbon nanomaterials for years. Here, the products obtained from carbon nanotubes and graphite after a mixed-acid treatment are carefully studied. Nearly identical carbon dot (Cdot) products with diameters of 3-4 nm are produced using this approach from a variety of carbon starting materials, including single-walled carbon nanotubes, multiwalled carbon nanotubes, and graphite. These Cdots exhibit strong yellow fluorescence under UV irradiation and shifted emission peaks as the excitation wavelength is changed. In vivo fluorescence imaging with Cdots is then demonstrated in mouse experiments, by using varied excitation wavelengths including some in the near-infrared (NIR) region. Furthermore, in vivo biodistribution and toxicology of those Cdots in mice over different periods of time are studied; no noticeable signs of toxicity for Cdots to the treated animals are discovered. This work provides a facile method to synthesize Cdots as safe non-heavy-metal-containing fluorescent nanoprobes, promising for applications in biomedical imaging.

Near-infrared light induced in vivo photodynamic therapy of cancer based on upconversion nanoparticles.

Upconversion nanoparticles (UCNPs) that emit high-energy photons upon excitation by the low-energy near-infrared (NIR) light are emerging as new optical nano-probes useful in biomedicine. Herein, we load Chlorin e6 (Ce6), a photosensitizer, on polymer-coated UCNPs, forming a UCNP-Ce6 supramolecular complex that produces singlet oxygen to kill cancer cells under NIR light. Excellent photodynamic therapy (PDT) efficacy is achieved in tumor-bearing mice upon intratumoral injection of UCNP-Ce6 and the followed NIR light exposure. It is further uncovered that UCNPs after PDT treatment are gradually cleared out from mouse organs, without rendering appreciable toxicity to the treated animals. Moreover, we demonstrate that the NIR-induced PDT based on UCNP-Ce6 exhibits a remarkably increased tissue penetration depth compared to the traditional PDT using visible excitation light, offering significantly improved treatment efficacy for tumors blocked by thick biological tissues. Our work demonstrates NIR light-induced in vivo PDT treatment of cancer in animals, and highlights the promise of UCNPs for multifunctional in vivo cancer treatment and imaging.

Endosomal pH-activatable poly(ethylene oxide)-graft-doxorubicin prodrugs: synthesis, drug release, and biodistribution in tumor-bearing mice.

Novel poly(ethylene oxide)-graft-doxorubicin (PEO-g-DOX) prodrugs with DOX covalently conjugated to PEO via a pH-sensitive hydrazone bond were developed. PEO-g-DOX conjugates could be readily prepared in the following steps: (i) anionic ring-opening copolymerization of ethylene oxide (EO) and allyl glycidyl ether (AGE) afforded functional PEO with controlled molecular weights, low polydispersities, and multiple pendant double bonds (PEO-g-allyl); (ii) conjugation of PEO-g-allyl with methyl mercaptoacetate, followed by treating with hydrazine hydrate, quantitatively transformed allyl into hydrazide groups (PEO-g-hydrazide); and (iii) DOX was covalently immobilized to PEO-g-hydrazide via acid-labile hydrazone bonds (PEO-g-DOX). Here on the basis of PEO-g-allyl(4.4) (M(n GPC) = 22 400, PDI = 1.19) and PEO-g-allyl(7.1) (M(n GPC) = 15 300, PDI = 1.16, the subscription refers to number of allyl groups per chain) two freely water-soluble PEO-g-DOX prodrugs with 2.9 and 3.6 DOX per molecule (denoted as PEO-g-DOX(2.9) and PEO-g-DOX(3.6), corresponding to drug loading content of 5.6 and 9.0 wt %, respectively) were obtained. The in vitro release studies confirmed much faster release of DOX at pH 5.0 and 6.0 than at pH 7.4. For example, approximately 16, 52, and 61% of drug were released in 22 h, and 23, 83, and 92% of drug were released in 120 h from PEO-g-DOX(2.9) at pH 7.4, 6.0 and 5.0, respectively. Notably, confocal laser scanning microscope (CLSM) observations revealed that DOX was released and delivered into the nuclei of RAW 264.7 cells following 24 h of incubation. MTT assays demonstrated that PEO-g-DOX(2.9) had pronounced cytotoxic effects to RAW 264.7, HeLa, and 4T1 breast tumor cells with IC(50) values of about 26.5, 42.5, and 32.0 µg DOX equiv/mL, whereas the corresponding polymer carrier PEO-g-hydrazide(4.4) was nontoxic. The In Vivo pharmacokinetics and biodistribution studies in mice showed that PEO-g-DOX(2.9) prodrugs had significantly prolonged circulation time and enhanced drug accumulation in the tumor as compared with free DOX. We are convinced that endosomal pH-activatable PEO-g-DOX prodrugs have tremendous potential for targeted cancer therapy.

Optimization of surface chemistry on single-walled carbon nanotubes for in vivo photothermal ablation of tumors.

Carbon nanotubes have shown great potential in various areas of biomedicine. Herein, we synthesize a series of amphiphilic polymers by anchoring polyethylene glycol (PEG) of different lengths at various densities on poly(maleic anhydride-alt-1-octadecene) (PMHC(18)). The blood circulation and biodistribution of those PEG-PMHC(18)-coated SWNTs in mice after intravenous injection are measured by an established Raman spectroscopy method. It is found that heavily PEGylated SWNTs with ultra-long blood circulation half-lives, although shows high uptake in the tumor, tend to accumulate in the skin dermis. A surface coating which affords SWNTs a blood half-life of 12-13 h appears to be optimal to balance the tumor-to-normal organ (T/N) uptake ratios of nanotubes in major organs. Using the selected SWNT conjugate, we then carry out a pilot in vivo photothermal therapy study and observe a promising cancer treatment efficacy. Our results highlight the importance of surface coating to the in vivo behaviors of nanomaterials in general and could provide guidelines to the future design of SWNT bioconjugates for various in vivo applications.