Two-Step Targeted Drug Delivery Via Proteinaceous Barnase-Barstar Interface and PLGA-Based Nano- Carrier

Victoria O. Shipunova (  viktoriya.shipunova@phystech.edu ) Moscow Institute of Physics and Technology National Research University: Moskovskij ziko-tehniceskij institut nacional'nyj issledovatel'skij universitet https://orcid.org/0000-0001-6361-1042 Elena N. Komedchikova Moscow Institute of Physics and Technology: Moskovskij ziko-tehniceskij institut nacional'nyj issledovatel'skij universitet Anna S. Sogomonyan Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry Russian Academy of Sciences: FBGUN Institut bioorganiceskoj himii im akademikov M M Semakina i U A Ovcinnikova Rossijskoj akademii nauk Polina A. Kotelnikova Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry Russian Academy of Sciences: FBGUN Institut bioorganiceskoj himii im akademikov M M Semakina i U A Ovcinnikova Rossijskoj akademii nauk Maxim P. Nikitin Moscow Institute of Physics and Technology: Moskovskij ziko-tehniceskij institut nacional'nyj issledovatel'skij universitet Sergey M. Deyev Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry Russian Academy of Sciences: FBGUN Institut bioorganiceskoj himii im akademikov M M Semakina i U A Ovcinnikova Rossijskoj akademii nauk


Introduction
Cancer is one of the most signi cant threats to humankind. In 2020 only, 19.1 million cases were registered, and this number is supposed to reach 28.4 million by 2040. It is the second most frequent death cause, and the contribution of cancer to the mortality rate continues to grow [1]. Conventional cancer therapy strategies suffer from a lack of selectivity and low drug e ciency and are frequently associated with side effects, including cardiac dysfunction, cytopenia, infection, diarrhea, vomiting, and others [2,3].
Chemotherapy-loaded nanoparticles capable of increasing the amount of therapeutic drug that reaches the tumor site and reducing the systemic toxicity provide encouraging solutions to the described problems. Moreover, drug encapsulation into the nanoparticle architecture can increase the bioavailability of the chemotherapeutic compound, extend the duration of action via bloodstream circulation prolongation, and can solve the problems associated with hydrophobicity and insolubility of drugs [4][5][6][7][8].
Several nanoparticle-based medications, such as PEGylated liposomal doxorubicin Caelyx® [9], and liposomal formulation of daunorubicin and cytarabine VYXEOS® [10], have already been approved by FDA for cancer treatment. However, despite the improved e ciency of such formulations, the delivery of nanoparticles occurs by passive transport through enlarged pores in the vascular endothelium of tumors known as the "enhanced permeability and retention (EPR)" effect [4]. However, it was shown that the EPR effect fails in some tumors and for some patients. Therefore, it is critical to develop different approaches for the delivery of nanoparticles to cancer cells [11]. One of the ways to implement targeted delivery is to modify the surface of nanoparticles with molecules binding certain cancer cell receptors. This is a rapidly developing branch of biomedicine that has already demonstrated several promising results in clinical trials [12,13]. Various proteins, such as antibodies, transferrin, EGF, lectins, as well as protein-nucleic acid complexes, aptamers, and small molecules like folic acid and sugars, are traditionally used for targeted drug delivery [14]. Currently, small synthetic polypeptides (scaffold molecules) emerge as the most promising targeting compounds due to their remarkable a nity, stability, ease of biotechnological production, and the absence of immunomodulation in vivo [14][15][16][17].
A pre-targeting concept implying two-step delivery of therapeutic compounds to tumor site is expected to provide signi cant systemic nanoparticle toxicity reduction and nanoparticle targeting abilities improvement [18]. This concept is based on the initial delivery of the rst targeting non-toxic compound to speci c cancer cells in a moderately high dose (thus realizing pre-targeting) followed by the delivery of a relatively small dose of a second toxic compound interacting with the rst one in a key&lock mode. A twostep drug delivery systems (DDS) offer a series of bene ts over standard one-step systems, such as 1) reduced toxicity for normal cells; 2) controlled penetration of toxin into the tumor; 3) improved drug biodistribution; 4) reduction of the required dose of the drug [19]. The disadvantages of the currently available two-step DDSs are due to the immunogenicity of the components, possible competition with molecules in the bloodstream, and the expensive and time-consuming biotechnological production in mammals of the components of such DDSs [20,21].
Here for the rst time, we used Barnase*Barstar pair as a platform for two-stage delivery of the oncotherapeutic compound. Ribonuclease Barnase and its natural inhibitor Barstar are small proteins (12 and 10 kDa) of bacterial origin that are not presented in mammals and possess an extremely high constant of binding (K aff = 10 14 M −1 ). We synthesized polymer PLGA nanoparticles and loaded them with the chemotherapeutic drug doxorubicin and the uorescent dye Nile Blue, and successfully modi ed the surface of nanoparticles with Barnase. The Barnase*Barstar interface was used as lego bricks to link the toxic PLGA nanoparticles with scaffold protein DARPin9_29 recognizing the tumor marker HER2 on the surface of cancer cells. DARPin9_29 was genetically fused with Barstar to obtain DARPin9_29-Barstar protein capable of speci cally targeting HER2-overexpressing cancer cells. We showed two-stage e cient labeling of HER2-overexpressing cancer cells with supramolecular structure PLGA-Barnase*DARPin9_29-Barstar self-assembled on the cell surface. We demonstrated the cytotoxicity of nanoparticles and more than tenfold therapeutic dose reduction versus free doxorubicin thus con rming the potential of this DDS for cancer treatment.

Materials And Methods
Nanoparticle synthesis PLGA nanoparticles were synthesized by the double emulsion "water-oil-water" method, followed by solvent evaporation, according to a modi ed procedure developed by us earlier [22]. The rst emulsion was obtained by adding 150 µL of an aqueous solution of doxorubicin hydrochloride at a concentration of 2 g/L to chloroform containing 300 µL of PLGA at a concentration of 40 g/L and 50 µL of Nile Blue at various concentrations, followed by sonication for 1 min at 40% amplitude and for 1 min at 60% amplitude using a 130 W ultrasonic disintegrator (Sonics) at +4°C. The second emulsion was created by mixing the rst emulsion with 3 mL of 5% polyvinyl alcohol solution in milliQ with the addition of 1 g/L chitosan oligosaccharide lactate. The solution was sonicated for 1 min at 40% amplitude and 1 min at 60% amplitude at +4°C. The resulting solution was incubated with slow shaking for chloroform evaporation, then washed three times in PBS by centrifugation, and resuspended in 300 µL of PBS PBS (137 mM NaCl, 2.7 mM KCl, 4.77 mM Na 2 HPO 4 ·2H 2 O, 1.7 mM KH 2 PO 4 , pH 7.4). The nal concentration of nanoparticles was determined by drying at 60°C and followed by weighing the dry residue.

Electron microscopy
Electron microscopy images of PLGA particles were obtained with a MAIA3 (Tescan) microscope at an accelerating voltage of 15 kV. The samples were deposited onto a silicon wafer and then dried in the air. The resulting images were processed using ImageJ software to obtain a particle size distribution.

Particle size and surface charge measurements
The hydrodynamic sizes and ζ-potential of nanoparticles were determined in PBS at 25°C using a Zetasizer Nano ZS (Malvern Instruments Ltd.) analyzer.

Nanoparticles conjugation with proteins
Barnase and Barstar were expressed and puri ed as described by us previously [23]. PLGA nanoparticles were covalently modi ed with Barnase or Barstar proteins using EDC and s-NHS as cross-linking agents through the formation of amide bonds between amino groups on the particle surface and protein carboxyl groups. 200 µg of protein was activated by a 15-fold molar excess of EDC and s-NHS in 0.1 M MES, pH 5.0 for 40 min at room temperature. Then protein was added to 1 mg of PLGA nanoparticles in 1) borate buffer -0.4 M H 3 BO 3 , 70 mM Na 2 B 4 O 7 ·10H 2 O, pH 8.0, 2) 0.1 M HEPES (4-(2-hydroxyethyl)-1piperazineethanesulfonic acid) pH 6.0, and sonicated. The mixture was incubated for at least 5 hours at room temperature, periodically treated in an ultrasonic bath, and the unbound protein was washed off by triple centrifugation for 5 minutes at 8000 g, nally resuspending the particles in PBS with 1% BSA.

Measurement of Barnase activity
The RNAse activity of Barnase was investigated by the method of the acid-insoluble precipitate [24]. The protein solution or solution of nanoparticles in 40 µL of buffer (0.125 M Tris-HCl, pH 8.5) was mixed with 160 µL of yeast RNA at a concentration of 2 g/L and incubated at 37° С for 15 min. The reaction was terminated by the addition of 200 µL of 0.625 N H 2 SO 4 and the mixture was incubated for 5 min at room temperature. Undigested RNA was separated by centrifugation at 14000 g for 15 min at room temperature. Optical density was measured at λ = 260 nm (OD260) corresponding to the concentration of free mononucleotides and proportional to the activity of the enzyme. The inhibition of Barnase RNAse activity was measured similarly: nanoparticles with Barstar were pre-incubated with 2.5 nM Barnase, and the enzymatic activity of the mixture was measured as described above.

Fluorescent microscopy
Excitation and emission spectra of PLGA nanoparticles were obtained using an In nite M100 Pro (Tecan) microplate reader. Nanoparticle suspension at a concentration of 10 µg/mL in 100 µL of PBS was placed in a 96-well at-bottomed plate. Excitation spectra were measured in the range from 350 to 675 nm (with the emission of 700 nm), emission spectra from 675 to 850 nm (excitation wavelength 650 nm).
The culture was grown in the thermostatic shaker at 25°C and 200 rpm overnight. Cells were harvested by centrifugation at 5000g for 15 min at 4 ºC. Cell pellets were resuspended in lysis buffer (20 mM Na-Pi, 300 mM NaCl, pH 7.4, 50 µg/mL lysozyme) and then sonicated on ice. Cellular debris was removed by centrifugation at 30000g at 4°C for 2 h. After the addition of imidazole (20 mM

Cell culture
Cell lines -human breast adenocarcinoma, SK-BR-3, and Chinese hamster ovary, CHO, were cultured in DMEM medium supplemented with 10% fetal bovine serum, penicillin-streptomycin, and 2 mM Lglutamine. Cells were incubated in a humidi ed atmosphere with 5% CO2 at 37°C. Cells were passaged when they reached 80-90% of the monolayer. To remove cells from the surface of the plastic 2 mM EDTA solution in PBS was used without trypsin addition (to avoid disruption of the integrity of cell receptors).
Cell lines were maintained in culture for no more than 2 months, after which they were replaced with fresh frozen lines. For cell counting, a Countess (Invitrogen) automatic cell counter was used. For this, 5 µL of 0.4% trypan blue, which stains only dead cells, was added to 5 µL of cell suspension. The solution was pipetted and added to the slides for the automatic cell counting.

Flow cytometry
To determine the e ciency of cells labeling with FITC-modi ed proteins, the cell suspension was washed with PBS, resuspended in 300 µL of PBS with 1% BSA at a concentration of 10 6 cells/mL. Cells were labeled with proteins in a nal concentration of 2 µg/mL, washed, and analyzed in FL1 channel (excitation laser -488 nm, emission lter -530/30 nm) using a BD Fortessa (BD) ow cytometer.
To determine the speci city of targeted nanoparticles, the cell suspension was washed in PBS, resuspended in 300 µL of PBS with 1% BSA at a concentration of 10 6 cells/mL.

Synthesis and characterization of polymer nanoparticles based on poly-(D, L-lactic-co-glycolic acid)
Poly-(D, L-lactic-co-glycolic acid) nanoparticles (PLGA) possessing both uorescent and cytotoxic properties were synthesized by the double water-oil-water emulsion method as shown in Figure 1.
Nile Blue ([9-(diethylamino)benzo[a]phenoxazin-5-ylidene] azanium sulfate, also known as Nile Blue A) was incorporated in the nanoparticles as a uorescent dye that allows to track the particles inside cells and use them for diagnostic purposes. This dye is successfully used in a wide range of biological applications, such as gel electrophoresis, staining of histological sections, labeling of neutral lipids and fatty acids, and visualization of cancer cells [27]. Nile Blue is a biocompatible dye with absorption and emission maxima in the near-infrared optical window (635 and 674 nm in water solution, respectively), which makes it optimal for labeling target cells in vitro and in vivo.
Doxorubicin was incorporated into the nanoparticles that allow using them for therapeutic applications.
Doxorubicin is an anthracycline antibiotic, that causes cell death by the interaction with DNA and inhibition of topoisomerase II, which leads to suppression of nucleic acids synthesis, and by the formation of free radicals, that destroy cellular membrane and biomolecules [28,29].
Nanoparticles were synthesized by the double emulsion "water-oil-water" method with subsequent evaporation of the solvent as shown in Figure 1.
The rst emulsion was obtained by the addition of water doxorubicin solution to the solution of PLGA and Nile Blue in chloroform, followed by a short sonication. The second emulsion was obtained by the addition of the rst emulsion to the polyvinyl alcohol solution containing 1 g/L of chitosan oligosaccharide lactate, followed by the second short sonication. After chloroform evaporation by the slow mixing, nanoparticles were centrifugated and resuspended in phosphate-saline solution (PBS).
The nanoparticles' morphology was studied by scanning electron microscopy (MAIA3 microscope, Tescan) at an accelerating voltage of 15 kV using an in-beam secondary electron detector. The received images (Fig. 2a) illustrate that synthesized PLGA nanoparticles are spherical monodisperse structures.
Image processing with ImageJ software shows that the average size and standard deviation of nanoparticles are 218 ± 59 nm (Fig. 2b). The hydrodynamic size of nanoparticles, measured by the dynamic light scattering method, was determined as 201 ± 38 nm ( The e cient incorporation of the uorescent dye Nile Blue was investigated by uorescence spectroscopy by measuring the excitation and uorescence emission spectra of nanoparticles. The excitation spectra were measured in the range from 350 to 675 nm (with emission at 700 nm). Four PLGA nanoparticles with different Nile Blue concentrations used in the synthesis were investigated. The excitation spectra (Fig. 2e) and emission spectra (Fig. 2f) demonstrate that the most effective Nile Blue concentration during the synthesis is 1.7 g/L, the further scaling up of Nile Blue concentration leads to the decrease in uorescence intensity. It is most probably caused by non-uorescent H-aggregates formation having an absorption shifted to the blue region of the spectrum.
The e cient incorporation of doxorubicin was investigated by uorescent spectroscopy on nanoparticles that do not contain Nile Blue. Nanoparticles were solved in DMSO and then uorescence was measured using a uorescence calibration curve for doxorubicin samples in the same solutions. The measurement of the uorescence of the samples showed that doxorubicin incorporation was 0.9 nmol doxorubicin per 1 mg of nanoparticles.
Barnase*Barstar protein interface for the targeted two-step delivery of PLGA particles to HER2-overexpressing cancer cells One of the central problems of modern chemotherapy is its relative non-speci city. The nanoparticle surface should be modi ed with targeting molecules in order to incorporate cancer cell targeting modalities into the nanoparticle structure and reduce non-speci c toxicity to normal non-transformed cells. To make this kind of modi cation universal for any target on the cell surface and include the possibility to "cancel the action on demand", we propose to mediate the interaction between toxic nanoparticles and molecules recognizing cancer cells using protein adaptors, the Barnase*Barstar protein pair. Barstar (10 kDa) is a natural inhibitor of bacterial ribonuclease Barnase (12 kDa) [23]. The N-and Cterms of both proteins are available for chemical conjugation and genetic engineering and are not located in the active site of both enzymes.
We used scaffold protein DARPin9_29 that recognizes the receptor HER2 on the surface of cancer cells with high a nity (K D = 3.8 nM) for the targeted delivery of synthesized polymer PLGA nanoparticles to cancer cells. This modular DDS based on PLGA nanoparticles, protein adaptors Barnase*Barstar, and scaffold proteins are schematically illustrated in Figure 3.
The surface of the nanoparticles was modi ed by one of the components of the pair - Figure 3 shows PLGA nanoparticles covalently modi ed with Barnase. During the pre-targeting process, Barstar- The e ciency of conjugation was measured by the enzymatic ability of conjugated nanoparticles to hydrolyze RNA by the commonly used method of the acid-insoluble precipitate [24]. First, the solution of conjugated PLGA nanoparticles was mixed with yeast RNA and incubated at 37°C to digest RNA. Then, the reaction was stopped by the addition of sulfuric acid, and uncleaved RNA was separated by centrifugation. The optical density of the solution corresponding to the concentration of free mononucleotides and proportional to the activity of the enzyme was measured. This value is proportional to the ribonuclease activity of the tested proteins or nanoparticles. The inhibition of RNAse activity of Barnase was measured similarly. Nanoparticles with Barstar were pre-incubated with Barnase, and the enzymatic activity of the mixture was measured as described above.
The enzymatic activity of free Barnase and Barstar proteins is shown in Figure 4a. The enzymatic activity of Barstar, namely the ability to inhibit Barnase, was investigated similarly by its pre-incubation with 2.5 nM of Barnase (green curve in Figure 4a) and measuring the enzymatic activity of the sample. As a positive control in the investigation of the enzymatic activity of PLGA nanoparticles conjugated with Barstar, a sample with Barstar at a concentration of 15 nM (+ Barnase 2.5 nM) was used.
We obtained three types of PLGA nanoparticles modi ed with Barnase by three different methods: i) carbodiimide conjugation at pH 8.0, ii) carbodiimide conjugation at pH 6.0, iii) non-covalent protein adsorption on the particle surface. Data presented in Figure 4b indicate that the highest e ciency of modi cation of PLGA nanoparticles is achieved during chemical conjugation at pH 6.0. Similar data were obtained for PLGA nanoparticles conjugated with Barstar: the highest inhibition of the Barnase activity is achieved for conjugates obtained at pH 6.0. Therefore, the possibility of obtaining functionally effective PLGA nanoparticles in terms of enzymatic activity with both Barnase and Barstar has been demonstrated.

Targeted delivery of polymer PLGA nanoparticles to the cells with oncomarker HER2 overexpression
One of the options, displayed in Figure 3 was employed to demonstrate the e ciency of the proposed scheme for the creation of targeted nanostructures for delivery to the cancer cells for their selective destruction. Namely, conjugates of PLGA nanoparticles with Barnase, PLGA-Bn, were obtained, and selfassembled on the cancer cell surface using Barstar fused with DARPin9_29. Thus, supramolecular structures PLGA-Bn*Bs-DARPin9_29 were assembled on the cell surface using pre-targeting concept via Bs-DARPin9_29 protein and subsequent binding with PLGA*Bn. These structures were delivered to the cells overexpressing receptor HER2.
For the in vitro experiments, we selected two cell lines with various levels of HER2 expression, namely SK-BR-3 and CHO cells. SK-BR-3 is a mammary adenocarcinoma cell line with overexpression of HER2 (about 10 6 receptors per cell), while CHO, Chinese hamster ovary cells, do not express any receptor of the EGFR family. Expression of HER2 receptor on these cells was con rmed by confocal microscopy (Fig. 5a) and by ow cytometry (Fig. 5b) by imaging cells with uorescence-labeled full-length antibody against HER2 -Trastuzumab-FITC. Also, the binding of DARPin9_29 was con rmed by cell labeling with DARPin9_29-FITC (Fig. 5b). Data from microscopy and cytometry assays presented in Figure 5a,b indicate that SK-BR-3 cells do express HER2 and are effectively labeled with full-length anti-HER2 antibody Trastuzumab and anti-HER2 scaffold protein DARPin9_29.
The functional activity of DARPin within the composition of fusion proteins with Barnase and Barstar -Bn-DARPin9_29 and Bs-DARPin9_29 was con rmed by ow cytometry (Fig. 5b)

Cytotoxicity of targeted supramolecular structures PLGA Bn*Bs DARPin9_29
Along with a uorescent dye, the synthesized PLGA polymer nanoparticles contain a chemotherapeutic drug, doxorubicin, which induces cell death via apoptosis. The cytotoxicity of PLGA-Bn*Bs-DARPin9_29 nanostructures was investigated by standard MTT test three days after adding the nanostructures in different concentrations to the HER2-overexpressing cells. The therapeutic effectiveness of targeted nanostructures was compared with free doxorubicin, which was added to the cells under similar conditions, as well as non-targeted PLGA nanoparticles loaded with doxorubicin.
The results of the cytotoxicity study of targeted PLGA nanoparticles with doxorubicin and free doxorubicin are presented in Fig. 5d, which shows the molar concentration of free doxorubicin and molar concentration of doxorubicin incorporated inside PLGA particles.
Half-maximal inhibitory concentration (IC50) was calculated for doxorubicin, PLGA, and PLGA-Bn*Bs-DARPin9_29. For free doxorubicin IC50 = 441 ± 61 nM, for doxorubicin in the nanoparticles IC50 = 42.7 ± 2.7 nM. Consequently, the incorporation of doxorubicin in the composition of targeted nanoparticles decreases its IC50 by 10.3 times. At the same time, cells exposed to non-targeted PLGA nanoparticles were not affected by the cytotoxic properties of PLGA and survived by more than 82 % even at the highest concentrations of PLGA, namely 1 g/L (Fig. 5d, violet curve). Hence, including a chemotherapeutic drug in the composition of polymer PLGA nanoparticles, assembled on the surface of the cancer cells via Barnase*Barstar interface, signi cantly decreases the concentration of chemotherapeutic drug doxorubicin, needed to receive the same cytotoxic effect.

Discussion
Polymer nanoparticles are the most promising vectors for targeted drug delivery due to their high biocompatibility, a wide spectrum of materials available for synthesis, and the ease of modi cation with molecules of different origins and functionality [30]. Among the wide range of natural and synthetic polymer materials for the design of therapeutic nanoparticles (such as protein-based polymers, polyphosphates, polyamides, polysaccharides, poly-lactic-co-glycolic acid) PLGA is the most popular polymer commonly used for biomedical and fundamental research application 25 . PLGA has been already approved by FDA for the therapeutic purposes and acts as a unique polymer for drug delivery [31,32].
PLGA is a co-polymer of fully biocompatible and biodegradable lactic and glycolic acids and has already demonstrated remarkable results in clinical trials as an excellent candidate for drug delivery and treatment [33,34].
To track the nanoparticles inside the organism, monitor their delivery and biodistribution, and use them for diagnostic tasks, we incorporated the uorescent dye, namely, Nile Blue into the nanoparticle structure.
Nile Blue is a uorescent dye from the benzophenoxazine family with high uorescence, high quantum yield, and excellent photostability [35]. The maximum excitation of the dye in dimethyl sulfoxide is 636 nm, and the maximum emission is 669 nm, thus entering the transparency window of biological tissues and making this dye promising for imaging applications in vivo [35]. Due to its lipophilic structure, Nile Blue was already used for several biological applications, e.g. for histology in vitro. Moreover, several in vivo studies have demonstrated the ability of this dye to accumulate in tumor cells after i.v. administration [27,36]. Despite the above-mentioned advantages and low cost, Nile Blue was unjustly underestimated in biology with a limited number of studies related to its usage.
Here we describe the development of a two-step drug delivery system, based on polymer PLGA nanoparticles possessing both diagnostic and therapeutic properties. The delivery of these nanoparticles to HER2-overexpressing cancer cells is realized via proteinaceous Barnase*Barstar interface and HER2recognizing scaffold protein DARPin9_29. The concept of targeted drug delivery suggests several advantages over standard chemotherapy, such as the decrease of the required dose of a drug, the improved drug penetration into the tumor, and the reduction of side effects [4]. However, the use of traditional targeting molecules for the therapeutics delivery, namely the use of monoclonal antibodies, with certain molecular pro les but also realizing their own diagnostic and therapeutic functions [22,25,39,42,49]. Here we used DARPin9_29 genetically fused with Barstar which showed highly speci c binding to receptor HER2 and allowed us to realize a two-stage delivery system based on Barnase*Barstar interface.  [50] ✓Both proteins were isolated from bacteria and are not represented in mammals [50].
✓ Covalent bonding is stronger than a nity interaction but is not reversible.
✓Both proteins were isolated from bacteria and modi ed by bioengineering and are not represented in mammals [71].
Several two-stage DDSs are currently in use and under development (see Table 1). As shown in Table 1, Barnase*Barstar protein pair outperforms other two-stage systems which makes it a unique tool for the design of multifunctional biomedical products. Barstar (10 kDa) is a natural inhibitor of bacterial ribonuclease Barnase (12 kDa) [23]. These proteins have an extremely high binding a nity (association constant K aff ~ 10 14 М -1 ) and fast interaction kinetics (rate constant of complex formation k on ~ 10 8 М -1 s -1 ), at the same time these proteins are not presented in mammals, which allows them to be used in blood ow without any interaction with endogenic components of blood [50,72]. In the present study, Barstar-DARPin9_29 was used as the rst component of the two-stage DDS and nanoparticles modi ed with Barnase served as the second component. It is also important to note, the lack of immunogenicity of all compounds used here -Barnase, Barstar, and Barstar-DARPin9_29.
Previously we showed the versatility of the Barnase*Barstar protein system for a wide range of applications including targeted delivery of both protein molecules, nanoparticles, and different supramolecular structures. In particular: i) the stability of the Barnase*Barstar protein complex under severe conditions (low pH, high temperature, and presence of chaotropic agents) was demonstrated that opens up more possibilities for using this system in any conditions both in vitro and in vivo [73]; ii) the successful labeling of HER2-overexpressing cancer cells in vitro with the self-assembled structures consisting of the magnetic particles and quantum dots using Barstar and scFv-Barnase-scFv construct (directed toward HER2 antigen) [21] and in vivo with radiolabeled 4D5 scFv-Barnase and 4D5 scFv-diBarnase [23] were shown; iii) a universal delivery system based on Barnase-Barstar and SiO 2 -binding peptide was developed [25]; iv) bispeci c antibodies against HER1 and HER2 antigens using 425scFv-Barstar and 4D5scFv-Barnase [74] were obtained and utilized for imaging of cancer cells with overexpression of these receptors [75]. All the described studies con rm the versatility of the Barnase*Barstar interface for the wide range of biological applications that require the self-assembly of different structures in different conditions. In this work, using Barstar-DARPin9_29 and Barnaseconjugated polymer PLGA nanoparticles loaded with uorescent dye Nile Blue and chemotherapeutic drug doxorubicin, we showed successful labeling and killing of HER2-overexpressing cells. The use of such two-step DDS allows decreasing the necessary dose of the doxorubicin needed to cause cancer cell death by one level of magnitude.

Conclusions
Here we report the versatile method of the two-stage drug delivery system (DDS) for theranostic applications based on Barnase*Barstar proteinaceous interface. The small size and high a nity constant of these proteins make them an excellent "molecular glue" for the design of different self-assembling structures based on various modules, where one component of this DDS is in the structure of one module (e.g., Barnase in the therapeutic module), and another system component in the structure of another module (e.g., Barstar in targeted DARPin module). This "lego" approach allows escaping such chemical

Ethics declarations
Ethics approval and consent to participate Not applicable.

Consent for publication
All authors read and approve the nal manuscript.
Availability of data and materials The datasets used and analysed during the current study are available from the corresponding author on reasonable request.   The scheme of the self-assembly of nanoparticles with scaffold polypeptides using protein interface Barnase*Barstar for the delivery to HER2-overexpressing cancer cells. The surface of nanoparticles is covalently modi ed with one of the adapters, Barnase, then the structure is assembled with Barstar, the second component of the pair, which is fused with targeting scaffold protein DARPin9_29. DARPin9_29 selectively recognizes receptor HER2 on the surface of cancer cells. This modular approach allows using the system for the assembly of different types of nanoparticles and target molecules. The enzymatic activity of PLGA conjugates with Barnase (purple bars) and Barstar (green bars). Data is presented for conjugates obtained at pH 8.0, pH 6.0, and by protein absorption on the particle surface.
Optical density (OD260) corresponded to the concentration of free mononucleotides was measured at a wavelength of 260 nm.

Supplementary Files
This is a list of supplementary les associated with this preprint. Click to download.