The effect of enhanced permeability and retention is often not sufficient for highly effective cancer therapy with nanoparticles, and the development of active targeted drug delivery systems based on nanoparticles is probably the main direction of modern cancer medicine. To meet the challenge, we developed polymer PLGA nanoparticles loaded with fluorescent photosensitive xanthene dye, Rose Bengal, and decorated with HER2-recognizing artificial scaffold protein, affibody ZHER2:342. The obtained 170 nm PLGA nanoparticles possess both fluorescent and photosensitive properties. Namely, under irradiation with the green light of 540 nm nanoparticles, they produced reactive oxygen species leading to cancer cell death. The chemical conjugation of PLGA with anti-HER2 affibody resulted in the selective binding of nanoparticles only to HER2-overexpressing cancer cells. HER2 is a receptor tyrosine kinase that belongs to the EGFR/ERbB family and is overexpressed in 30% of breast cancers, thus serving as a clinically relevant oncomarker. However, the standard targeting molecules such as full-size antibodies possess serious drawbacks, such as high immunogenicity and the need for mammalian cell production. We believe that the developed affibody-decorated targeted photosensitive PLGA nanoparticles will provide new solutions for ongoing problems in cancer diagnostics and treatment, as well in cancer theranostics.
Nanoparticle-based chemotherapy is considered to be an effective approach to cancer diagnostics and therapy in modern biomedicine. However, efficient tumor targeting remains a great challenge due to the lack of specificity, selectivity, and high dosage of chemotherapeutic drugs required. A two-step targeted drug delivery strategy (DDS), involving cancer cell pre-targeting, first with a first nontoxic module and subsequent targeting with a second complementary toxic module, is a solution for decreasing doses for administration and lowering systemic toxicity. To prove two-step DDS efficiency, we performed a direct comparison of one-step and two-step DDS based on chemotherapy loaded PLGA nanoparticles and barnase*barstar interface. Namely, we developed and thoroughly characterized the two-step targeting strategy of HER2-overexpressing cancer cells. The first targeting block consists of anti-HER2 scaffold polypeptide DARPin9_29 fused with barstar. Barstar exhibits an extremely effective binding to ribonuclease barnase with Kaff = 1014 M−1, thus making the barnase*barstar protein pair one of the strongest known protein*protein complexes. A therapeutic PLGA-based nanocarrier coupled to barnase was used as a second targeting block. The PLGA nanoparticles were loaded with diagnostic dye, Nile Blue, and a chemotherapeutic drug, doxorubicin. We showed that the two-step DDS increases the performance of chemotherapy-loaded nanocarriers: IC50 of doxorubicin delivered via two-step DDS was more than 100 times lower than that for one-step DDS: IC50 = 43 ± 3 nM for two-step DDS vs. IC50 = 4972 ± 1965 nM for one-step DDS. The obtained results demonstrate the significant efficiency of two-step DDS over the classical one-step one. We believe that the obtained data will significantly change the direction of research in developing targeted anti-cancer drugs and promote the creation of new generation cancer treatment strategies.
The development of non-invasive photothermal therapy (PTT) methods utilizing nanoparticles as sensitizers is one of the most promising directions in modern oncology. Nanoparticles loaded with photothermal dyes are capable of delivering a sufficient amount of a therapeutic substance and releasing it with the desired kinetics in vivo. However, the effectiveness of oncotherapy methods, including PTT, is often limited due to poor penetration of sensitizers into the tumor, especially into solid tumors of epithelial origin characterized by tight cellular junctions. In this work, we synthesized 200 nm nanoparticles from the biocompatible copolymer of lactic and glycolic acid, PLGA, loaded with magnesium phthalocyanine, PLGA/Pht-Mg. The PLGA/Pht-Mg particles under the irradiation with NIR light (808 nm), heat the surrounding solution by 40 °C. The effectiveness of using such particles for cancer cells elimination was demonstrated in 2D culture in vitro and in our original 3D model with multicellular spheroids possessing tight cell contacts. It was shown that the mean inhibitory concentration of such nanoparticles upon light irradiation for 15 min worsens by more than an order of magnitude: IC50 increases from 3 µg/mL for 2D culture vs. 117 µg/mL for 3D culture. However, when using the JO-4 intercellular junction opener protein, which causes a short epithelial–mesenchymal transition and transiently opens intercellular junctions in epithelial cells, the efficiency of nanoparticles in 3D culture was comparable or even outperforming that for 2D (IC50 = 1.9 µg/mL with JO-4). Synergy in the co-administration of PTT nanosensitizers and JO-4 protein was found to retain in vivo using orthotopic tumors of BALB/c mice: we demonstrated that the efficiency in the delivery of such nanoparticles to the tumor is 2.5 times increased when PLGA/Pht-Mg nanoparticles are administered together with JO-4. Thus the targeting the tumor cell junctions can significantly increase the performance of PTT nanosensitizers.
Hybrid multimodal nanoparticles, applicable simultaneously to the noninvasive imaging and therapeutic treatment, are highly demanded for clinical use. Here, Fe-Au core-satellite nanoparticles prepared by the method of pulsed laser ablation in liquids were evaluated as dual magnetic resonance imaging (MRI) and computed tomography (CT) contrast agents and as sensitizers for laser-induced hyperthermia of cancer cells. The biocompatibility of Fe-Au nanoparticles was improved by coating with polyacrylic acid, which provided excellent colloidal stability of nanoparticles with highly negative ζ-potential in water (−38 ± 7 mV) and retained hydrodynamic size (88 ± 20 nm) in a physiological environment. The ferromagnetic iron cores offered great contrast in MRI images with r2 = 11.8 ± 0.8 mM−1 s−1 (at 1 T), while Au satellites showed X-ray attenuation in CT. The intravenous injection of nanoparticles enabled clear tumor border visualization in mice. Plasmonic peak in the Fe-Au hybrids had a tail in the near-infrared region (NIR), allowing them to cause hyperthermia under 808 nm laser exposure. Under NIR irradiation Fe-Au particles provided 24.1 °C/W heating and an IC50 value below 32 µg/mL for three different cancer cell lines. Taken together, these results show that laser synthesized Fe-Au core-satellite nanoparticles are excellent theranostic agents with multimodal imaging and photothermal capabilities.
The aim of this work is to develop a 3D cell culture model based on cell spheroids for predicting the functional activity of various compounds in vivo. Agarose gel molds were made using 3D printing. The solidified agarose gel is a matrix consisting of nine low-adhesive U-shaped microwells of 2.3 3.3 mm for 3D cell spheroid formation and growth. This matrix is placed into a single well of a 12-well plate. The effectiveness of the cell culture method was demonstrated using human ovarian carcinoma SKOVip-kat cells stably expressing the red fluorescent protein Katushka in the cytoplasm and overexpressing the membrane-associated tumor marker HER2. The SKOVip-kat cell spheroids were visualized by fluorescence microscopy. The cell concentration required for the formation of same-shape and same-size spheroids with tight intercellular contacts was optimized. To verify the developed model, the cytotoxicity of the targeted immunotoxin anti-HER2 consisting of the anti-HER2 scaffold DARP 9_29 and a fragment of the Pseudomonas aeroginosa exotoxin, DARP-LoPE, was studied in 2D and 3D SKOVip-kat cell cultures. The existence of a difference in the cytotoxic properties of DARP-LoPE between the 2D and 3D cultures has been demonstrated: the IC50 value in the 3D culture is an order of magnitude higher than that in the monolayer culture. The present work describes a universal tool for 3D cultivation of mammalian cells based on reusable agarose gel molds that allows for reproducible formation of multicellular spheroids with tight contacts for molecular and cell biology studies.
The extreme aggressiveness and lethality of many cancer types appeal to the problem of the development of new-generation treatment strategies based on smart materials with a mechanism of action that differs from standard treatment approaches. The targeted delivery of nanoparticles to specific cancer cell receptors is believed to be such a strategy; however, there are no targeted nano-drugs that have successfully completed clinical trials to date. To meet the challenge, we designed an alternative way to eliminate tumors in vivo. Here, we show for the first time that the targeting of lectin-equipped polymer nanoparticles to the glycosylation profile of cancer cells, followed by photodynamic therapy (PDT), is a promising strategy for the treatment of aggressive tumors. We synthesized polymer nanoparticles loaded with magnetite and a PDT agent, IR775 dye (mPLGA/IR775). The magnetite incorporation into the PLGA particle structure allows for the quantitative tracking of their accumulation in different organs and the performing of magnetic-assisted delivery, while IR775 makes fluorescent in vivo bioimaging as well as light-induced PDT possible, thus realizing the theranostics concept. To equip PLGA nanoparticles with targeting modality, the particles were conjugated with lectins of different origins, and the flow cytometry screening revealed that the most effective candidate for breast cancer cell labeling is ConA, a lectin from Canavalia ensiformis. In vivo experiments showed that after i.v. administration, mPLGA/IR775–ConA nanoparticles efficiently accumulated in the allograft tumors under the external magnetic field; produced a bright fluorescent signal for in vivo bioimaging; and led to 100% tumor growth inhibition after the single session of PDT, even for large solid tumors of more than 200 mm3 in BALB/c mice. The obtained results indicate that the mPLGA/IR775 nanostructure has great potential to become a highly effective oncotheranostic agent.
The conventional methods of treating cancer with chemo- and radiotherapy present plenty of serious problems, such as low therapeutic index and high systemic toxicity. The advanced cancer treatment strategies utilize nanoformulations of drugs that can enter a tumor due to the enhanced permeability and retention (EPR) effect. However, EPR fails in the treatment of several human diseases. Mainstream biomedical studies are focused on creating the drugs that would enter the tumor with higher effectiveness and require smaller doses for administration. A two-stage drug delivery system is an encouraging alternative solution. At first, the primary, non-toxic targeting module is delivered to the tumor cells, followed by injection of the second complementary targeting module at a considerably lower dose, thus decreasing systemic toxicity. To meet the challenge, we have developed a two-stage drug delivery system (DDS), mediated by the high-affinity binding of the Barnase*Barstar protein pair. Barnase and Barstar act as lego bricks linking the first and the second modules on the surface of the cancer cell. Barnase (12 kDa) is a natural ribonuclease from Bacillus amyloliquefaciens, while Barstar (10 kDa) is its natural inhibitor. The Barnase*Barstar is one of the strongest known protein*protein complexes with Kaff = 1014 M−1 exhibiting extraordinarily stability in severe conditions. Artificial scaffold polypeptide DARPin9_29 genetically fused with Barstar served is a first module of the developed two-step DDS. DARPin9_29 (14 kDa) specifically recognizes the tumor marker HER2 overexpressed on human breast cancer cells. As a second module, a therapeutic nano-cargo was developed based on fluorescent polymer PLGA nanoparticles loaded with diagnostic Nile Blue dye and the chemotherapeutic drug doxorubicin. This nano-PLGA structure was covalently coupled to Barnase. We showed two-stage efficient labeling of HER2-overexpressing cancer cells using the first non-toxic module DARPin9_29-Barstar and the second toxic nano-module PLGA-Barnase. We demonstrated the doxorubicin-induced cytotoxicity of this two-step DDS based on polymer nanoparticles and proteinaceous Barnase-Barstar interface and showed more than 10-fold therapeutic dose reduction versus free doxorubicin. We believe that the developed two-step DDS based on PLGA nano-cargo and protein interface will promote the creation of new-generation cancer treatment strategies.
Oncotheranostic nanoagents are powerful artificial compounds that significantly outperform traditional antitumor therapies. Among the wide variety of nanoagents, polymer nanoparticles have proven to be one of the most successful platforms for translation into the clinic. Here we show the development of an oncotheranostic nanoagent based on poly(lactic-co-glycolic acid) (PLGA) with red/green light dual-activated diagnostic/therapeutic properties for targeted photodynamic therapy of HER2-positive aggressive breast cancer. PLGA particles were loaded with red light-activated Nile Blue dye with pronounced solvatochromic properties for diagnostic applications and with green light-activated photodynamic sensitizer Rose Bengal for photodynamic therapy. The nanoparticles were decorated with anti-HER2 antibodies during the synthesis without chemical conjugation which is very important for further large-scale biotechnological production. In vitro and in vivo studies proved the red-light mediated HER2-specific imaging and green-light induced cytotoxicity of obtained anti-HER2 PLGA. Interestingly, these particles fluoresce only when internalized into cells, which minimizes background signals until the complete elimination from the organism, thus facilitating real-time diagnostics. We have shown that such particles allow efficient and selective localization of the HER2-positive primary tumor node, as well as metastases, and achieve complete remission in BALB/c Nu/Nu mice with the HER2-positive xenografts using the single session of photodynamic therapy.
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