Introduction

Nanotechnology represents a new field of science which has special potential to treat a broad variety of human diseases. Nanoparticles have favorable properties for detecting, imaging, and treatment of disease such as controlling and targeting drug release, improving solubility, reducing dose-related toxicity, and better absorption. Also, some other benefits are as follows: reduction of frequency of drug administration and the cost of treatment and [1] better cancer diagnosis and molecular imaging [2]. Many different platforms have been developed, including synthetic nanomaterials and naturally bionanomaterials such as viral nanoparticles (VNPs). VNPs are one of the biggest groups of bionanoparticles which originated from plant, animal, or bacterial viruses [3]. Biocompatibility and biodegradability of VNPs offer advantages over synthetic nanoparticles. Capsids of the filamentous or isometric viruses with multidimensional structures are commonly used as bionanoparticles in pharmaceutical nanotechnology [4, 5]. VNPs derived from plant viruses are less likely to interact with the mammalian system and make adverse effects [6–8]. The unique structures, regular geometric shapes, dynamic self-assembling systems and monodisperse structure of non-pathogenic plant viruses can be useful in the delivery of proteins or other molecules and strongly influence on the conjugation between the therapeutic agent or other molecules and nanoparticles [9]. The larger surface area of filamentous viruses provides more potential binding and acceptor sites for functionalization, compared to isometric particles. Filamentous particles also present ligand in a more efficient manner. A rod-shaped particle may in theory interact with a larger number of binding sites on the cell surface, thus increasing targeting sensitivity and specificity [10].

The filamentous potato virus X (PVX) particles are flexible rods 515 × 12 nm consisting of 1270 identical 25-kDa coat protein (CP) subunits. PVX is a plant pathogenic virus of the family Alphaflexiviridae and the order Tymovirales. It is a type of species of the genus Potexvirus [11].

Breast cancer alone is expected to account for 29 % of all new cancers among women [12]. Early detection often allows more effective treatment. It is well-known that about 20–30 % of all breast cancers are HER2/neu-positive. HER2-positive breast cancers tend to be more aggressive than the other types of breast cancer [13]. Monoclonal antibody Herceptin (Trastuzumab) is a humanized recombinant anti-HER2/neu mAb that is inhibiting cell proliferation and signaling transduction. Herceptin can especially bind to the membrane region of HER2/neu with a high affinity [14].

Targeted adenovirals with HER2/neu have enhanced the transduction efficiently of adenovirus. The conjugation of super-paramagnetic iron oxides nanoparticles with Herceptin in MRI have been improved their targeting of the specific receptor in cells and magnetization [15].

VLP nanoparticles have been intensively investigated as a novel drug carrier due to their extensive advantages such as: good biocompatibility, biodegradability, and non-toxic properties [8, 9]. Therefore, VLP nanoparticles are urgently needed to improve the efficiency of anticancer drug delivery. In this paper, we aim at preparing Herceptin conjugated PVX (PVX-HER) to improve tumor-targeted drug delivery. Herceptin was covalently coupled to PVX with utilizing the chemical linker (EDC/sulfo-n-hydroxysuccinimide (sulfo-NHS)). Morphology, size, and conjugation efficiency of prepared PVX-HER particles were characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Western blot, ELISA, Zeta potential and transmission electron microscopy.

The result indicates that the PVX-HER nanoparticles have efficiency compared to free Herceptin in SK-OV-3 and SK-BR-3 cells by flow cytometry. This research demonstrates that the obtained PVX-HER particles can be used for potential targeting delivery of anticancer drugs.

Materials and methods

PVX was spread in Nicotiana glutinosa, and leaves were harvested 2 weeks after mechanical inoculation with 5 μg of purified PVX particles. The PVX was purified as follows: 100 g of leaves were homogenized in a standard blender using 2 vol. of 0.1 M phosphate buffer (pH 8, 0.1 M, 2-mercaptoethanol 0.02, 10 % ethanol) and was filtered through two to three layers of cheesecloth. Plant material was centrifuged at 7800g for 20 min and supernatant collected. After adding NaCl 0.2 M and 4 % (w/v) PEG (MW 8000), the solution was centrifuged at 7800g for 30 min. The pellet was resuspended in 1 % Triton X 100 and 0.05 M phosphate buffer followed by centrifugation at 7800g for 10 min. The supernatant was ultracentrifuged in a 30 % sucrose gradient at 72,500g for 150 min. The pellet was resuspended in 0.05 M phosphate buffer (pH 7.2). The virus concentration in plant extract was determined by UV/vis spectroscopy. Being assured of the quality of the purified virus by SDS-PAGE, the virus was stored in −20 °C [16].

PVX-Herceptin conjugation (PVX-HER)

Herceptin (IV infusion), EDC [1-ethyl-3-(3-dimethyl amino propyl) carbodiimide hydrochloride] and sulfo-NHS were purchased from Hoffmann-La Roche, Merck (851,007) and Thermo (24,510), respectively [17].

The overall steps of PVX-Herceptin conjugation and formation are illustrated in Fig. 1. To optimize conjugation reactions, EDC/sulfo-NHS was added to reaction buffer (0.5 M NaCL, 0.1 M MES, pH 6) at different concentrations and time courses as indicated in the corresponding figures (30 min, 2 h, 4 h, 6 h, and overnight). In the processes of binding PVX to Herceptin with chemical linkers, different concentrations of EDC/sulfo-NHS with a ratio of 1:2.5 were used in this experiment [18]. The concentrations used in this study are shown in Table 1. The first step of reaction was quenched by 2-mercaptoetanol and Biogel P-10 Column (BioRad) as removal of unconjugated-free linkers.

Fig. 1
figure 1

The conjugation process of viral nanoparticles with monoclonal antibody Herceptin using EDC/sulfo-NHS in phosphate buffer, after each step samples were purified by gel filtration columns

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Table 1 Evaluation of different combination of cross-linker. EDC and sulfo-NHS by zeta potential values of PVX-HER conjugate in different cross-linker concentrations [18]
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In the second step of conjugation, Herceptin antibodies were added at ten times more concentrated than PVX virus [10] to sodium phosphate buffer (pH 7.5, 0.1 M NaCL) at different incubation times (2, 4, 6 h, and overnight). PVX-Herceptin nanoparticles were purified by a Biogel P-10 column (BioRad). The number of Herceptin molecules per version was determined by measuring the absorbance at 490 nm.

The efficiency of conjugation

The efficiency of conjugation between PVX and monoclonal antibody Herceptin (PVX-HER) was investigated by different methods including sandwich ELISA, SDS-PAGE, Western blot, and Zetasizer.

ELISA

PVX-HER nanoparticles were analyzed by sandwich ELISA assay in 96-well plates (Nunk Denmark). The bottom of each well was coated with PVX-specific antibodies (DSMZ, PV-0027) diluted 1:1000 in coating buffer (0.05 M carbonate-bicarbonate, pH 9.6). Plates were then blocked by PBS buffer containing 2 % PVP and 1.5 % BSA at 37 °C for 90 min. After blocking, samples of PVX, Herceptin, and PVX-HER were added to wells and incubated at 37 °C for 90 min. Triplicate samples and blanks needed to be run with each plate to ensure accuracy. Free Herceptin was cleared out with washing buffer (0.05 % (v/v) Tween-20 in PBS). The secondary antibody (goat polyclonal secondary antibody to human IgG-HRP) manufactured by Abcam Co. (ab97175) was used at a dilution of 1:2000 and incubated for 1 h. TMB Reagent (3,3′,5,5′-tetramethylbenzidine, T0565 Sigma) was added, and the reaction terminated after sufficient color development by adding 100 μl of stop solution (2 M H2SO4) to each well. Then, the optical density was measured at 450 nm.

Denaturing gel electrophoresis

SDS gel electrophoresis was carried out to analyze the conjugation of PVX to Herceptin. Ten micrograms of the protein samples were analyzed on 8–12 % biorad gels (Ready Gel Tris–HCl-161-1394). PVX-HER particles, Herceptin, PVX, and a protein marker (prestained protein ladder, Thermo; 2616) were run at 100 V for 45 min. For Western blotting, protein was transferred from gels to nitrocellulose membrane in a transfer buffer at 350 mA for 90 min. Then, Ponceau S staining solution was used to detect protein bands on PVDF or nitrocellulose membranes. The membranes were blocked in 4 % nonfat dry milk at 37 °C for 2 h. Next, one of the membranes was blotted with PVX antibody (DSMZ, PV-0027) at 4 °C overnight, followed by staining with goat polyclonal secondary antibody to rabbit IgG-HRP manufactured by Abcam Co. (ab6721) for 120 min. Another nitrocellulose membrane incubated with goat polyclonal secondary antibody to human IgG-HRP (1:15,000) for 2 h (ab97175). ECL chemiluminescent substrate kit was applied to detect Western blots according to manufacturer protocol (GE Healthcare, Advance Western Blotting Detection Kit, RPN 2235). Protein bands were then visualized by exposure the membrane to photographic film for 1 min.

Measurement of surface charge by Zetasizer

Zeta potential measurements were carried out using a ZEN 3600 (Malvern Instruments Co., USA) for PVX, Herceptin, and PVX-HER (1.25 ml of 0.1 mg ml−1 solutions) with five measurements, each including ten runs.

Characterization of morphology by transmission electron microscopy (TEM)

Drops of PVX-HER were placed on carbon-coated copper grids (10, 0.1 mg ml−1) and allowed to absorb for 5 min. Then, it was washed three times with 100 μL of deionized water and negatively stained with 1 % (w/v) uranyl acetate for 2 min. Samples were analyzed by FEI Tecnai T20G2 transmission electron microscope. TEM analysis was used to observe the filamentous integrity nanostructures (515 × 12) when conjugation processes were carried out.

Flow cytometry

SK-OV-3 and SK-BR-3 cells were cultured in RPMI-1640 medium supplemented with 10 % fetal bovine serum (FBS), 1 % l-glutamine, penicillin (100 unit/ml) and streptomycin (100 μg/ml). Cells were maintained at 37 °C in a humidified 5 % CO2 incubator. The culture medium was changed every other day, and the cells were passaged when they reached 80 to 90 % confluency. SK-BR-3 (2 × 105) and SK-OV-3 (1 × 105) cells were seeded on a 12-well plate. After 24 h, each cell was infected with 10 μg free Herceptin (H10), 20 μg free Herceptin (H20), 40 μg free Herceptin (H40), 80 μg free Herceptin (H80), conjugation of PVX-HER that contains 10 μg Herceptin (V-H10), and 20 μg Herceptin (V-H20). The average of triplicates for each concentration was then plotted to free virus and control cells. Two separate plates were designed for each SK-OV-3 and SK-BR-3 cells and were analyzed after 24 and 48 h. The cells were stained by PI and Annexin V-FITC Kit (BMS500 FI/100, eBioscience) and then washed three times with PBS. Then, 300 μl of PBS was added to perform flow cytometric analysis with FACScan, and a total of 10,000 events per sample were collected.

Results

In this study, PVX particles were purified from infected Nicotiana glutinosa plants, yielding 0.5–1.0 mg pure PVX per 1 g infected leaves. PVX concentration in plant extracts was determined by UV/visible spectroscopy (2.97 mg−1 ml−1 cm−1). The purity of the virus was confirmed based on the A260:A280 ratio (1.2). The purity of PVX was determined by a single protein band at 27 kDa by SDS-PAGE, which is the expected size of PVX coat protein [19]. Then, we investigated several cross-linkers in the primary steps to find an optimal cross linker. Herceptin was covalently coupled to carboxylate groups on the external surface of PVX by EDC/sulfo-NHS methods, and the molar ratio of Herceptin to PVX was 10:1 (HER:PVX). The optimal cross-linker concentration (2:5, 4:10, 8:20) and incubation time (30 min, 2 h, 4 h, 6 h, and overnight) to conjugate PVX to Herceptin were determined by Western blotting and Zetasizer measurement. The results obtained by Western blotting analysis indicated that the appropriate ratio concentration of EDC/sulfo-NHS is 4:10 (Fig. 2a, b).

Fig. 2
figure 2

a The zeta potential of the PVX-HER particles was analyzed by Zetasizer to detect the optimal concentration for EDC/sulfo-NHS. The zeta potential for EDC/sulfo-NHS at the molar ratios of 2:5 (1), 4:10 (2), and 8:20 (3), was compared to the zeta potential for Herceptin and virus, 1.48 and −21.4 mV, respectively. b Western blot analysis using human antibody to determine the optimal concentration for EDC/sulfo-NHS. The ratio of EDC/sulfo-NHS was 2:5 (line 1); 4:10 (line 2) and 8:20 (line 3)

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Zetasizer exhibited a zeta potential of −1.48 and −21.4 mV for Herceptin and PVX, respectively. A different zeta potential of PVX-HER, depending on the concentration of EDC/sulfo-NHS (1/2.5), was obtained as shown in Table 1. Graphs obtained from Zetasizer apparatus are in Fig. 2a.

Successful conjugation of PVX-Her was investigated by SDS-PAGE and Western blotting. An equal amount of protein was separated by 8–12 % Tris-HCl SDS-PAGE (biorad) and transferred to a nitrocellulose membrane. After the transfer, the membrane was stained with Ponceau S staining to investigate protein size. As expected, our staining showed that Herceptin is composed of two 25 and 55 kDa protein bands corresponding to light and heavy chains, respectively. PVX showed a protein band of 27 kDa as coat protein and finally PVX-HER conjugate represented an extra band of approximately 83 kDa. The conjugation of PVX coat protein (27 KDa) and heavy chain of Herceptin (55 KDa) are shown in Fig. 3b. We further investigated these results by Western blotting. The Western blot analysis of nitrocellulose membrane exposed to human antibody showed a protein band of HER with a molecular weight of 55 kDa and PVX-HER conjugate with molecular weight of 83 kDa. The Western blot analysis of nitrocellulose membrane exposed to PVX specific antibody showed a protein band with molecular weight of 27 kDa and PVX-HER conjugate band with molecular weight of 83 kDa (Fig. 3a).

Fig. 3
figure 3

a Analysis of PVX-HER conjugation by Western blotting. PVX specific antibody and HRP-conjugated specific anti human antibody are illustrated in left and right image, respectively. Single protein band (27 kDa) of purified PVX was found in the line 1, pure Herceptin was found in the line 2 that no evidence of protein band related to PVX specific antibody was observed, 83 kDa of PVX-HER conjugate was found in line 3. Line 4 was pure PVX that no evidence of band with HRP-conjugated specific anti human antibody was observed, Herceptin alone (55 kDa) in line 5, PVX-HER conjugate was found in line 6 that the protein band of heavy antibody with molecular weight of 55 kDa which was much more less than 83 kDa was also observed. b Conjugations and changes in protein molecular weight in SDS-PAGE gels. Using Panceau S staining; Line 1 Pure PVX coat protein subunits with molecular weight of 27 kDa, lines 2–4 monoclonal antibody Herceptin consists of heavy and light chains with molecular weights of 55 and 25 kDa, respectively; line 5 PVX- HER conjugate and formation of protein band with molecular weight of 83 kDa. Line M page ruler prestained protein ladder (Thermo; 2616)

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Another approach to approve the conjugation was obtained by enzyme-linked immunosorbent assay (ELISA). In this method, we set up a sandwich ELISA assay where the PVX antibody was used to coat the plate and an anti-human conjugate was used to detect conjugated PVX-HER bound to the anti PVX antibody. Our ELISA results approved the conjugation between PVX and Herceptin. Also, PVX-HER had higher absorption compared to HER and virus controls alone (Fig. 5a).

An important aspect of our conjugation strategy was to maintain the integrity of the PVX particles. To investigate the nanoparticle structure of our conjugate, we applied TEM. TEM images showed that the filamentous shape of PVX particles remained intact after the conjugation of PVX and Herceptin (Fig. 4a, b).

Fig. 4
figure 4

Analysis of integrity of natural PVX and PVX-HER particles by transmission electron microscope (TEM). a Filamentous structure of PVX (650 × 12 nm) was observed by TEM. b PVX-HER conjugates show filamentous structure similar to PVX after conjugation

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Following successful conjugation of PVX-Herceptin conjugation, we investigated whether PVX-HER particles had a better cytotoxicity against HER-positive cell lines SK-OV-3 and SK-BR-3. Both cell lines were incubated in two different concentrations of PVX-HER particles (10 and 20 μg) and four different concentrations of Herceptin (10, 20, 40, and 80 μg) as well as free PVX as control (Fig. 5b). Following 24 h treatment with PVX-HER particles, the average percentage of apoptotic cells in SK-BR-3 was 45.77 %, and 70.18 % in V-H10 (consist 10 μg Herceptin) and V-H20 (consist 20 μg Herceptin), respectively, while the corresponding numbers for the equal amounts of Herceptin alone (H10 and H20) were 19.9 and 24.2 %, respectively. After 48 h, the percentage of apoptotic cells in SK-BR-3 was 27.72 % in V-H10 and 41.58 % in V-H20 (Fig. 6) that is showing an increase compared to H10 (13.2 %) and H20 (18.9 %).

Fig. 5
figure 5

a Analysis of PVX-HER conjugation by sandwich ELISA. Absorbance at a wavelength of 450 nm (Y-axis) indicates the absorption of PVX (V), Herceptin (H) alone, and PVX-HER (V-H) (X-axis), respectively. b Analysis of potential apoptotic activity of PVX-HER particles compared to unconjugated Herceptin. Percentage of apoptotic cells (Y-axis) of SK-BR-3 cells after 24 (blue) and 48 h (red): H10, H20, H40, H80, V-H10, and V-H20 (from left to right), c Percentage of cell death (Y-axis) with apoptosis of SK-OV-3 cells after 24 (blue) and 48 h (red): H10, H20, H40, H80, V-H10, and V-H20 (from left to right)

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Fig. 6
figure 6

Representative results of flow cytometric analysis of apoptosis in SK-BR-3 treated with PVX-Herceptin particles. a Analysis of the H20 and b V-H20 with Annexin V-FITC (FL1) and propidium iodide (FL3). The lower right quadrant (Q4) contains the early apoptotic (Annexin V+/PI−) population and upper right (Q2) quadrant contains the late apoptotic/necrotic (Annexin V+/PI+) population

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After 24 h, SK-OV-3 apoptotic cells induced by V-H10 and V-H20 were 37.26 and 54.75 %, respectively, that is showing an increase compared to H10 (16.2 %) and H20 (21.9 %). After 48 h, apoptosis induced by viral nanoparticles was 21.25 % in V-H10, 34.32 % in V-H20, 10.12 % in H10, and 15.6 % in H20 (Fig. 5c).

Discussion and conclusion

The era of targeted therapies has emerged to reduce the cost of treatment and minimize unwanted side effects. Nanotechnology offers novel opportunities for disease treatment in human health care because they can be tailored to partition cargos between diseases and healthy cells. Different kinds of materials are being produced and used; these include synthetic and natural nanomaterials, e.g., protein cages and capsids formed by viruses.

Selection of each class of nanomaterials depends on their advantages and disadvantages. As a nano carrier, VNPs have many favorable properties such as self assembly, stability, [3] produced large quantities in short time [6], biodegradable, and non-toxicity in mammals [20].

On the other hand, in case of immunogenicity, it can even be beneficial in cancer treatment in a better way to target immune systems to the tumor site.

VNPs are excellent platforms to use in biomedicine. It is known that nanoparticles are used to deliver cargo to vimentin-positive (cancer) cell. VNPs offer this by interacting with surface vimentin on tumor cells [21]. Also, VNPs are smaller than liposomes, and consequently, they are more ideal for cell targeting and uptake and enhance tissue penetration compare to liposomes [22].

Recent studies indicated that filamentous nanomaterials have superior pharmacokinetic and tumor-homing properties. It has been reported that filamentous plant VNPs has enhanced tumor homing and tissue penetration compared to isometric VNPs. This is mainly due to increased surface of the filamentous particle compared to Isometric particle. The larger surface area of filamentous VNPs provides more potential binding sites, compared to isometric VNPs thus increasing targeting capacity and specificity [10].

Nanoparticles have drawn great attention as targeted imaging and therapeutic agents. Also, Herceptin is a monoclonal antibody applied in treatment of HER2-positive breast cancers. Since it is an expensive drug, Herceptin-linked nanoparticles in cancer therapy reduce doses of drugs required for treatment and enhance the stability of Herceptin by slowing the decomposition of the drug. The main goal of this study was to investigate the potential use of PVX particles as a Herceptin coated nanoparticle.

Although adenovirus-associated toxicity has been reported in animal cells [8], adenovirus and EDC/sulfo-NHS have been used for HER-adenovirus conjugate [17]. We used EDC/sulfo-NHS as a cross-linker in our study. EDC/sulfo-NHS activates carboxyl groups to conjugate to the amino group. In this study, EDC/sulfo-NHS cross-linker was used to conjugate the heavy chain of HER with the PVX coat protein in a two-steps process. First, the PVX was incubated with cross-linker for 4 h; then in the second step, PVX-linker and Herceptin were incubated for 2 h. Protein assay proved that around 1000 molecules were conjugated on PVX-HER complex. All the concentrations and incubation times were optimized based on the results obtained by Zetasizer and Western blotting. We were able to detect an 83 kDa band. Mobility shift in the bands was representing the conjugation of Herceptin heavy chain and PVX coat protein. In sandwich ELISA, PVX-specific antibody coated as capture and also human antibodies added to the final step of process led to recognition of the complexes composed of only PVX-HER molecules.

The results derived from SDS-PAGE showed the combination of purified PVX coat protein (27 kDa) with heavy chain of antibody Herceptin (55 kDa) and the formation of protein (83 kDa). Western blot using PVX-specific antibody and Herceptin confirmed the results.

The average surface charge value of HER-PVX was −7.05, confirming that PVX-HER was well conjugated. The value obtained by Zetasizer for HER-PVX complex was between PVX (−21.4) and HER2 (−1.48). Hence, higher positive surface charge on PVX-HER was due to presence of HER. The conjugation of HER and PVX produced a value of −7.05 (in 4:10 ratio) for surface charge of HER-PVX compared to −21.4 PVX. These results also confirmed that HER was conjugated on the surface of PVX.

The morphologies of HER-PVX were observed by TEM. The TEM images revealed no integrity changes in the HER-PVX complex filamentous structure, indicating that cross-linkers and chemicals have no destructive effects on the viral structure.

Plant virus nanoparticles have not shown to be pathogenic to animal cells. This together with the low-cost method of purification and the maintenance of filamentous structure after cross linking play an important role in targeted cancer therapy.

In the last step, PVX-HER nanoparticles were examined to assess cytotoxicity against two HER2-positive cell lines, SK-OV-3, and SK-BR-3. Different doses of HER and PVX-Herceptin were used to treat both cell lines and analyze the cytotoxic effects by flow cytometry. Nanoparticles concentrations equal to 10 and 20 μg/ml of Herceptin were used in this study. SK-BR-3 and SK-OV-3 cells were demonstrated a great change in cell apoptosis caused by PVX-Herceptin compared to free Herceptin in the similar concentration. Cell death by apoptosis was examined after 24 and 48 h in two different Her 2-positive cell lines. SK-BR-3 apoptotic cells death appeared to be 2.8 (V-H10), 2.69 (V-H20) times more than using free Herceptin in 24 h; and 3.47 (V-H10), 4.91 (V-H20) times more than in free Herceptin 48 h. SK-OV-3 apoptotic cells demonstrate 1.39 (V-H10) and 1.45 (V-H20) times more than free Herceptin in 24 h; and 1.63 (V-H10), 1.47 (V-H20) times more than free Herceptin after 48 h.

VLP are highly dynamic structure and depicted as rigid closed shells that can undergo various structural transition. In recent years, a few VLP drug conjugations have been developed. The VLP nanoparticles were targeted to the endolysosomal compartment of the cells without the need for diffusion of the cell membrane with cell-specific targeting agent [23]. In this study, we lay the foundation to use PVX as a carrier for drug delivery. The goal of this study was to evaluate drug efficiency in two HER2-positive cell lines. The PVX has been showed advantages in cells penetration and tumor homing because of the shape and positive charges. Consistent with the last finding, cationic nanoparticles exhibit higher vascular permeability and also enhanced tumor homing properties compared to their anionic counterparts.

PVX-HER conjugation offers an advantage over free HER. At two dosages, PVX-HER is more effective cytotoxic agent toward SK-OV-3 and SK-BR-3 cells than free Herceptin. After 24 and 48 h, cells were effectively killed when treated with the PVX-HER conjugation at this concentration.

This is the first report on the successful conjugation of PVX viral nanoparticles with monoclonal antibody for treatment of cancer cells. Metathorax-conjugated dendrimers have been used in the treatment of patients with breast cancer. Dendritic structure-related toxicity and the position of the complex within the cell were also examined [24]. The results of this study revealed that the rate of cell death caused by PVX-Herceptin nanoparticles was significantly higher compared to that of Herceptin alone.

The study proved that monoclonal antibodies have the potential to conjugate with filamentous plant viruses such as PVX. The results of this study can reduce the economic costs of treatment with Herceptin. We hope that this finding will be applied extensively in the future.

The manuscript does not contain clinical studies or patient data.