Zera-3Ag protein nanoparticles for preventing and treating BVD and IBR as well as preparation method and application of Zera-3Ag protein nanoparticles

Abstract

The invention is suitable for the technical field of protein vaccines, and provides Zera-3Ag protein nanoparticles for preventing and treating BVD and IBR as well as a preparation method and application of the Zera-3Ag protein nanoparticles, and the Zera-3Ag protein nanoparticles comprise E2-1b-Zeraa fusion protein, E2-1d-Zeraa fusion protein and gD-Zeraa fusion protein; the amino acid sequences of the E2-1b-Zera fusion protein, the E2-1d-Zera fusion protein and the gD-Zera fusion protein are respectively shown as SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6 in a sequence table. After animals are immunized by the Zera-3Ag protein nano-particles, an effective immune protection effect can be generated, and the Zera-3Ag protein nano-particles have a good prevention and control application prospect on bovine viral diarrhea and bovine rhinotracheitis.

Description

Technical Field

[0001] This invention belongs to the field of protein vaccine technology, and particularly relates to a Zera-3Ag protein nanoparticle for the prevention and treatment of BVD and IBR, its preparation method and application. Background Technology

[0002] Bovine viral diarrhea virus (BVDV) and infectious bovine rhinotracheitis virus (IBRV) are pathogens that are prevalent in cattle herds worldwide. Their infection not only causes respiratory, reproductive, and digestive diseases such as bovine viral diarrhea (BVD) and bovine infectious rhinotracheitis (IBR), but also leads to decreased production performance and reproductive disorders in cattle herds, resulting in serious economic losses to the livestock industry.

[0003] BVDV belongs to the genus Menthaviridae in the family Flaviviridae. Based on genotype, it can be divided into BVDV-1, BVDV-2, and BVDV3. The main BVDV subtypes circulating in my country are BVDV 1a, BVDV 1b, and BVDV 1d. Its genome consists of approximately 12.3 kb of positive-sense single-stranded RNA, with the coding sequence as follows: Npro, capsid protein (C), Erns, E1, E2, p7, NS2 / NS3, NS4A, NS4B, NS5A, and NS5B. The E2 glycoprotein contains the major antigenic determinant, and its N-terminus is a major target of humoral immune responses and can induce neutralizing antibodies. The C-terminus of its extracellular domain has receptor binding and membrane fusion functions, which are key to mediating the binding of BVDV to host cell receptors (such as CD46 and LDL-R). In addition, recent studies have found that E2 can also bind to pattern recognition molecules involved in complement activation, making the virus more sensitive to complement lysis activity, thus indicating that E2 is an excellent candidate antigen for developing BVDV subunit vaccines.

[0004] While inactivated and attenuated vaccines widely used in clinical practice can alleviate viral outbreaks to some extent, their weak cross-protection, insufficient duration of immunity, and safety risks make them ineffective in controlling persistent infection and transmission, failing to fully meet the disease control needs of modern large-scale aquaculture systems. Therefore, developing novel vaccines with safe, efficient, and broad-spectrum protective effects has become a crucial direction for BVD and IBR prevention and control. In recent years, nanotechnology-based vaccine delivery systems, with their high-density antigen loading, strong immunogenicity, and excellent biocompatibility, have provided a new pathway for the development of multivalent vaccines. In-depth research on the immunizing effects of different nanoparticles on BVDV and IBRV vaccines, addressing the synergistic prevention and control needs, has significant theoretical and practical value. Summary of the Invention

[0005] The purpose of this invention is to provide a Zera-3Ag protein nanoparticle for preventing and treating BVD and IBR, aiming to solve the problems mentioned in the background art.

[0006] To address the aforementioned problems, the present invention provides a Zera-3Ag protein nanoparticle for preventing and treating BVD and IBR, comprising E2-1b-Zera fusion protein, E2-1d-Zera fusion protein, and gD-Zera fusion protein; the amino acid sequences of the E2-1b-Zera fusion protein, E2-1d-Zera fusion protein, and gD-Zera fusion protein are shown in the sequence listing SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:6, respectively.

[0007] Another object of the present invention is to provide a gene encoding the aforementioned Zera-3Ag protein nanoparticles for the prevention and treatment of BVD and IBR, said gene comprising the E2-1b-Zera gene, the E2-1d-Zera gene, and the gD-Zera gene; the nucleotide sequences of the E2-1b-Zera gene, the E2-1d-Zera gene, and the gD-Zera gene are shown in the sequence listing SEQ ID NO:1, SEQ ID NO:3, and SEQ ID NO:5, respectively.

[0008] Another object of the present invention is to provide a recombinant expression vector containing the above-mentioned genes, specifically including an E2-1b-Zera expression vector, an E2-1d-Zera expression vector, and a gD-Zera expression vector; wherein the E2-1b-Zera expression vector contains the E2-1b-Zera gene; the E2-1d-Zera expression vector contains the E2-1d-Zera gene; and the gD-Zera expression vector contains the gD-Zera gene.

[0009] Furthermore, the recombinant expression vector is a baculovirus expression vector.

[0010] Another object of the present invention is to provide a method for preparing the above-mentioned Zera-3Ag protein nanoparticles, comprising the following steps: E2-1b-Zera expression vector, E2-1d-Zera expression vector and gD-Zera expression vector were constructed using the genes with nucleotide sequences as shown in the sequence listing SEQ ID NO:1, SEQ ID NO:3 and SEQ ID NO:5, respectively. Recombinant Bacmid was prepared by transforming E2-1b-Zera expression vector, E2-1d-Zera expression vector and gD-Zera expression vector into host cells, respectively. The recombinant Bacmid was identified by PCR amplification; The correctly identified Bacmid was transfected into Sf9 cells for expression, resulting in E2-1b-Zera fusion protein, E2-1d-Zera fusion protein and gD-Zera fusion protein; E2-1b-Zera fusion protein, E2-1d-Zera fusion protein and gD-Zera fusion protein were mixed to obtain Zera-3Ag protein nanoparticles.

[0011] Furthermore, the molar ratio of the E2-1b-Zera fusion protein, the E2-1d-Zera fusion protein, and the gD-Zera fusion protein is (0.5-1.5):(0.5-1.5):(0.5-1.5).

[0012] Another object of the present invention is to provide the use of the above-mentioned Zera-3Ag protein nanoparticles, or the above-mentioned gene, or the above-mentioned recombinant expression vector in the preparation of drugs or vaccines for the prevention and treatment of bovine viral diarrhea and / or bovine rhinotracheitis.

[0013] Another object of the present invention is to provide a drug or vaccine for the prevention and treatment of bovine viral diarrhea and / or bovine rhinotracheitis, comprising a pharmaceutically acceptable carrier and the aforementioned Zera-3Ag protein nanoparticles.

[0014] This invention provides a Zera-3Ag protein nanoparticle for the prevention and treatment of BVD and IBR. By introducing Zera, which originates from the proline-rich N-terminal domain of γ-zein, large, degradation-resistant nanoparticles with high stability can be formed. These Zera-3Ag protein nanoparticles can be formulated into nanoparticle vaccines that are non-toxic to mammals and pose no biosafety threat. Furthermore, they elicited strong humoral and cellular immune responses in mice and cattle after immunization, indicating that the Zera-3Ag protein nanoparticles exhibit strong neutralizing activity in vivo after immunization of mice and cattle. Attached Figure Description

[0015] Figure 1 This is a schematic diagram illustrating the construction of the recombinant expression vector provided in an embodiment of the present invention.

[0016] Figure 2 Electron micrographs provided for embodiments of the present invention.

[0017] Figure 3 The images show SDS-PAGE and Western Blot diagrams of the expression and purification of E2-1b-Zera fusion protein (A), E2-1d-Zera fusion protein (B), and gD-Zera fusion protein (C) provided in the embodiments of the present invention.

[0018] Figure 4 The image shows the serum IgG detection results of mice immunized with Zera-3Ag protein nanoparticles provided in this embodiment of the invention.

[0019] Figure 5 The graph shows the results of spleen lymphocyte stimulation index detection in mice after immunization with Zera-3Ag protein nanoparticles provided in this embodiment of the invention.

[0020] Figure 6 The figure shows the results of the expression level analysis of the cytokine IFN-γ in the serum cell supernatant of mice after immunization with the Zera-3Ag protein nanoparticles provided in the embodiments of the present invention.

[0021] Figure 7 The figure shows the results of the analysis of the expression level of the cytokine TNF-α in the serum cell supernatant of mice after immunization with the Zera-3Ag protein nanoparticles provided in the embodiments of the present invention. Detailed Implementation

[0022] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0023] This invention employs the Baculovirus Expression Vector System (BEVS), a highly efficient recombinant protein expression platform using baculoviruses as vectors and insect cells as hosts, widely used in vaccine development and the development of therapeutic proteins. BEVS enables high-level expression of large, complex recombinant proteins and possesses post-translational modification capabilities characteristic of eukaryotic cells. Furthermore, BEVS is non-pathogenic to mammals, safe to operate, suitable for use in routine laboratories, and exhibits high biosafety. Simultaneously, insect cells are suitable for high-density growth and industrial-scale production. Several commercial vaccines are already produced based on the BEVS platform, demonstrating its maturity and feasibility in vaccine production and providing a powerful tool for modern vaccine and biopharmaceutical development.

[0024] To investigate a bivalent Zera nanoparticle vaccine against BVDV and IBRV, this invention prepared Zera nanoparticles containing the E2 protein of BVDV 1b and 1d types and the gD protein of IBRV using a baculovirus expression system. These nanoparticles were then mixed at a specific molar ratio to prepare Zera-3Ag nanoparticles. Immunization of BALB / c mice and evaluation of serum IgG and cytokine levels showed that the prepared Zera-3Ag protein nanoparticle vaccine had good immunogenicity.

[0025] Specifically, in one embodiment of the present invention, a Zera-3Ag protein nanoparticle for preventing and treating BVD and IBR is provided, comprising E2-1b-Zera fusion protein, E2-1d-Zera fusion protein, and gD-Zera fusion protein; wherein, the E2-1b-Zera fusion protein, E2-1d-Zera fusion protein, and gD-Zera fusion protein have a Zera sequence added to the N-terminus and a His6 tag added to the C-terminus; the amino acid sequences of the above-mentioned E2-1b-Zera fusion protein, E2-1d-Zera fusion protein, and gD-Zera fusion protein are shown in SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:6, respectively; the amino acid sequence of the above-mentioned Zera sequence is shown in SEQ ID NO:8, and the nucleotide sequence is shown in SEQ ID NO:7.

[0026] In another embodiment of the present invention, a gene encoding the aforementioned Zera-3Ag protein nanoparticles for preventing and treating BVD and IBR is also provided, the gene comprising the E2-1b-Zera gene, the E2-1d-Zera gene, and the gD-Zera gene; the nucleotide sequences of the E2-1b-Zera gene, the E2-1d-Zera gene, and the gD-Zera gene are shown in the sequence listing SEQ ID NO:1, SEQ ID NO:3, and SEQ ID NO:5, respectively.

[0027] In another embodiment of the present invention, a recombinant expression vector containing the above-mentioned genes is also provided, specifically including an E2-1b-Zera expression vector, an E2-1d-Zera expression vector, and a gD-Zera expression vector; the E2-1b-Zera expression vector contains the E2-1b-Zera gene; the E2-1d-Zera expression vector contains the E2-1d-Zera gene; and the gD-Zera expression vector contains the gD-Zera gene. Preferably, the above-mentioned recombinant expression vector is a baculovirus expression vector, and its promoter type is the polh promoter.

[0028] In another embodiment of the present invention, a method for preparing the above-mentioned Zera-3Ag protein nanoparticles is also provided, comprising the following steps: S1. Construct E2-1b-Zera expression vector, E2-1d-Zera expression vector and gD-Zera expression vector using the genes with nucleotide sequences as shown in the sequence listing SEQ ID NO:1, SEQ ID NO:3 and SEQ ID NO:5, respectively. S2. Recombinant Bacmid was prepared by transforming E2-1b-Zera expression vector, E2-1d-Zera expression vector and gD-Zera expression vector into host cells, respectively. S3. Perform PCR amplification and identification of the recombinant Bacmid; S4. Transfect the correctly identified Bacmid into Sf9 cells for expression to obtain E2-1b-Zera fusion protein, E2-1d-Zera fusion protein and gD-Zera fusion protein; S5. Mix the E2-1b-Zera fusion protein, E2-1d-Zera fusion protein and gD-Zera fusion protein to obtain Zera-3Ag protein nanoparticles.

[0029] Specifically, the method for transfecting Sf9 cells for expression is as follows: correctly identified Bacmid is transfected into Sf9 cells for expression, P0 generation virus is harvested by centrifugation, and Sf9 cells are infected again. The supernatant is harvested as P1 generation virus. P2 and P3 generation viruses are prepared in the same way. After 3 days of infection with P3 generation virus, cells are enriched, lysed and purified to obtain E2-1b-Zera fusion protein, E2-1d-Zera fusion protein and gD-Zera fusion protein.

[0030] Preferably, the host cell is Escherichia coli DH10 (BAC) competent cells; the molar ratio of the E2-1b-Zera fusion protein, E2-1d-Zera fusion protein and gD-Zera fusion protein is (0.5-1.5):(0.5-1.5):(0.5-1.5), more preferably 1:1:1.

[0031] In another embodiment of the present invention, the use of the above-mentioned Zera-3Ag protein nanoparticles, or the above-mentioned gene, or the above-mentioned recombinant expression vector in the preparation of drugs or vaccines for the prevention and treatment of bovine viral diarrhea and / or bovine rhinotracheitis is also provided.

[0032] In another embodiment of the present invention, a drug or vaccine for preventing and treating bovine viral diarrhea and / or bovine rhinotracheitis is also provided, comprising a pharmaceutically acceptable carrier and the aforementioned Zera-3Ag protein nanoparticles.

[0033] Example 1: This example provides a method for obtaining the target gene and constructing an expression vector, as detailed below: The gene sequences of bovine viral diarrhea virus (BVDV)-E2-1b (GenBank ID: AGM75780.1), BVDV-E2-1d (GenBank ID: MF166858.1), and bovine rhinotracheitis virus (IBRV)-gD (GenBank ID: QB159528.1) were selected and cloned into the baculovirus expression vector pFast1. A gp67 signal peptide and a Zera sequence were introduced at the N-terminus of the genes, and a 6×His tag was added at the C-terminus. BamHI and KpnI restriction sites were added to both ends of the constructed sequences. Codon optimization for the insect baculovirus expression system was performed on all genes to obtain the E2-1b-Zera expression vector, E2-1d-Zera expression vector, and gD-Zera expression vector. The constructed gene maps are shown below. Figure 1 As shown.

[0034] Example 2: This example provides a method for preparing recombinant rod-like particles, detecting protein expression, and collecting viruses, as detailed below: The E2-1b-Zera, E2-1d-Zera, and gD-Zera expression vectors prepared in Example 1 were transformed into *E. coli* DH10 (Bac) competent cells, respectively. Positive strains were obtained through blue-white screening, and positive Bacmids were extracted and named rBM-E2-1b-Zera, rBM-E2-1d-Zera, and rBM-gD-Zera, respectively. Sf9 cells were transfected using Beyotime's LipoInsect™ transfection reagent. After 96 h of transfection, the cell supernatant was collected and named rBV-E2-1b-Zera, rBV-E2-1d-Zera, and rBV-gD-Zera, respectively. Virus-infected cells were loaded onto a 200-mesh copper grid, stained with phosphotungstic acid, and observed morphologically under an electron microscope. Figure 2 As shown.

[0035] Example 3: This example provides a method for the amplification, purification, and detection of recombinant baculovirus, as detailed below: In pre-coated 6-well plates, rBV-E2-1b-Zera was added to each well with an MOI of 0.1, and two generations of amplification were performed to obtain relatively stable P3 generation baculovirus. Sf9 cells infected with P3 generation rBV-E2-1b-Zera, rBV-E2-1d-Zera, and rBV-gD-Zera were enriched by centrifugation at 6000×g and 4℃ for 10 min. The cells were resuspended in HM buffer, sonicated at 100 W for 10 min, and then slowly added to a 20%-40%-60% sucrose gradient solution. The cells were centrifuged at 80000×g for 2 h at 4℃, and the intermediate phase was collected to obtain E2-1b-Zera fusion protein, E2-1d-Zera fusion protein, and gD-Zera fusion protein. The expression of recombinant baculovirus was identified by Western blotting using anti-Anti-6×His as the primary antibody. The results are as follows. Figure 3 As shown.

[0036] In addition, the obtained E2-1b-Zera fusion protein, E2-1d-Zera fusion protein and gD-Zera fusion protein are mixed in a molar ratio of 1:1:1 to obtain Zera-3Ag protein nanoparticles.

[0037] Example 4: This example is a mouse immunization experiment, as detailed below: Thirty-six 6-week-old SPF-grade female BALB / c mice were randomly divided into three groups of 12 mice each, with an average weight of 20 ± 2 g. Immunization was performed via subcutaneous multi-site injection at 0, 14, and 28 days. The Zera-3Ag group (Zera-3Ag protein nanoparticles prepared in Example 3) received a total immunization dose of 30 μg, combined with M903 adjuvant; the commercially available dual vaccine group received a total immunization dose of 100 μL; and the PBS control group received 100 μL of PBS. Blood was collected from the tail at 0, 14, 28, 35, and 42 days. On 35 and 42 days, tail blood collection and spleen lymphocyte proliferation experiments were performed on four mice from each group.

[0038] Serum antibody levels were detected using ELISA at different time points (7 d, 14 d, 21 d, 28 d, 35 d, and 42 d) after mouse immunization. Serially diluted IgG standards and 1:100,000 diluted mouse serum samples were incubated in ELISA plates at 37°C for 1 h, washed, and HRP-labeled goat anti-mouse IgG secondary antibody was added, followed by incubation at 37°C for 1 h. After washing, 90 μL of TMB chromogenic solution was added, and the reaction was carried out at 37°C for 30 min. The reaction was terminated by adding 50 μL of 2 M H₂SO₄, and the absorbance was measured at 450 nm (OD450 nm). IgG antibody kinetic curves were plotted, and the results are shown below. Figure 4 As shown in the figure, the Zera-3Ag protein nanoparticles provided in this embodiment of the invention can effectively enhance the IgG immune response and induce more durable antibody levels.

[0039] Splenic lymphocyte proliferation assay: On days 35 and 42 post-immunization, three mice from each group were randomly selected and sacrificed by dislocation. Splenic tissue was collected from the mice, and the cells were gently ground in spleen lymphocyte separation medium (Beijing Dakowei Biotechnology Co., Ltd.) to prepare a single-cell suspension. The cells were centrifuged at 800×g for 30 min to separate the cells into layers. The lymphocyte layer was aspirated and resuspended and washed with RPMI-1640 complete medium. Press 1×10 6 Cells were seeded at a density of [number] cells / mL into 96-well plates, with Zera-3Ag protein nanoparticles (final concentration 5 μg / mL) added to each well. A commercially available vaccine group (dual vaccine) and a negative control group (PBS) were also established. After culturing at 37°C and 5% CO2 for 42 h, 10 μL of CCK-8 solution was added, and incubation continued at 37°C for another 4 h. OD values ​​were measured at 450 nm, and the lymphocyte proliferation index (SI) was calculated. SI = (OD value of experimental group - OD value of blank control group) / (OD value of negative control group - OD value of blank control group). Results are shown below. Figure 5 As shown, the SI in the PBS group remained at a low level at 35 and 42 days without significant change (p>0.05); the SI of the commercial vaccine Dual vaccine increased significantly at 42 days compared to 35 days (p<0.05), indicating that it could activate T cell responses late after immunization, but the proliferation level was limited. The Zera-3Ag group showed significant T cell proliferation capacity at 35 days, which was further enhanced at 42 days, indicating that the Zera-3Ag protein nanoparticles can more effectively stimulate T cell activation and play a key role in promoting T cell proliferation.

[0040] Analysis of Cytokine Levels in Mouse Serum Cell Supernatant: The secretion levels of cytokines in mouse serum cell supernatant were detected using a commercially available ELISA kit. First, mouse IFN-γ and TNF-α capture antibodies were coated onto 96-well ELISA plates and incubated overnight at 4°C, followed by blocking with 5% BSA for 1 h. Diluted serum was then added, and the plates were incubated at 37°C for 2 h, washed, and then biotin-labeled detection antibodies were added sequentially. The plates were incubated at 37°C for 30 min, and then washed to remove non-specific bindings. 90 μL of TMB chromogenic buffer was added, and the plates were reacted at 37°C in the dark for 15 min. Stop solution was then added, and the absorbance was measured at 450 nm. The concentrations of IFN-γ and TNF-α were calculated using a standard curve. Results are shown below. Figures 6-7 As shown, there was no significant difference in IFN-γ levels among the groups at 21 days post-immunization (p>0.05). At 42 days post-immunization, the IFN-γ levels in the Zera-3Ag group were higher than those at 21 days and also higher than those in the PBS group (p<0.05). The results indicate that the Zera-3Ag protein nanoparticles provided in this embodiment of the invention help promote cell-mediated immune responses.

[0041] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A Zera-3Ag protein nanoparticle for the control of BVD and IBR, characterized in that, The Zera-3Ag protein nanoparticles comprise an E2-1b-Zera fusion protein, an E2-1d-Zera fusion protein and a gD-Zera fusion protein; the amino acid sequences of the E2-1b-Zera fusion protein, the E2-1d-Zera fusion protein and the gD-Zera fusion protein are shown in SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6 respectively.

2. A gene encoding Zera-3Ag protein nanoparticle for controlling BVD and IBR as claimed in claim 1, characterized by, The genes comprise an E2-1b-Zera gene, an E2-1d-Zera gene and a gD-Zera gene; the nucleotide sequences of the E2-1b-Zera gene, the E2-1d-Zera gene and the gD-Zera gene are shown in SEQ ID NO: 1, SEQ ID NO: 3 and SEQ ID NO: 5 respectively.

3. A recombinant expression vector comprising the gene of claim 2, characterized in that, The E2-1b-Zera expression vector comprises the E2-1b-Zera gene; the E2-1d-Zera expression vector comprises the E2-1d-Zera gene; and the gD-Zera expression vector comprises the gD-Zera gene.

4. The recombinant expression vector of claim 3, wherein, The recombinant expression vector is a baculovirus expression vector.

5. A method for preparing Zera-3Ag protein nanoparticles as described in claim 1, characterized in that, The method comprises the following steps: The E2-1b-Zera expression vector, the E2-1d-Zera expression vector and the gD-Zera expression vector are constructed by using the genes with the nucleotide sequences shown in SEQ ID NO: 1, SEQ ID NO: 3 and SEQ ID NO: 5 respectively; The E2-1b-Zera expression vector, the E2-1d-Zera expression vector and the gD-Zera expression vector are transformed into host cells respectively to prepare recombinant Bacmids; PCR amplification is performed on the recombinant Bacmids for identification; The Bacmids that pass the identification are transfected into Sf9 cells for expression to obtain the E2-1b-Zera fusion protein, the E2-1d-Zera fusion protein and the gD-Zera fusion protein; The E2-1b-Zera fusion protein, the E2-1d-Zera fusion protein and the gD-Zera fusion protein are mixed to obtain the Zera-3Ag protein nanoparticles.

6. The method of claim 5, wherein the Zera-3 Ag protein nanoparticle is prepared by, The molar ratio of the E2-1b-Zera fusion protein, the E2-1d-Zera fusion protein and the gD-Zera fusion protein is (0.5-1.5):(0.5-1.5):(0.5-1.5).

7. Use of the Zera-3Ag protein nanoparticles of claim 1, or the genes of claim 2, or the recombinant expression vector of claim 3 in the preparation of a medicine or vaccine for preventing and treating bovine viral diarrhea and / or bovine rhinotracheitis.

8. A medicament or vaccine for the prevention and / or treatment of bovine viral diarrhea and / or bovine rhinotracheitis, comprising a pharmaceutically acceptable carrier, characterized in that, The Zera-3Ag protein nanoparticles of claim 1 are also included.

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