Annular single-stranded DNA delivery vector and application thereof

Abstract

The invention discloses a circular single-stranded DNA delivery carrier and application thereof, and belongs to the technical field of pharmaceutical preparations, the circular single-stranded DNA delivery carrier comprises ionizable cationic lipid, a lipid polyethylene glycol conjugate, a sterol compound and 1-stearoyl-2-oleoyl lecithin; in terms of the total mole number of total lipid in the annular single-stranded DNA delivery carrier being 1, the molar percentage of the ionizable cationic lipid is 25%-60%, the molar percentage of the lipid polyethylene glycol conjugate is 1.0%-3.5%, the molar percentage of the sterol compound is 20%-50%, and the molar percentage of the 1-stearoyl-2-oleoyl lecithin is 5%-30%. The delivery vector delivers the annular single-stranded DNA under the condition that the N / P ratio is (4-10): 1, shows excellent transfection efficiency, and has wide application prospects in the field of gene therapy.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical formulation technology, specifically relating to a circular single-stranded DNA delivery vector and its applications. Background Technology

[0002] Gene therapy is a novel treatment method based on genetic engineering technology that has developed rapidly in recent years. Gene therapy directly targets the root cause of disease—the abnormal gene itself—rather than merely alleviating symptoms. It typically involves introducing exogenous normal genes or therapeutic genes into target cells to correct or compensate for the disease caused by defective or abnormal genes, thereby achieving a therapeutic effect. In gene therapy, suitable vectors are essential for delivering genetic material to target tissues / cells. Common delivery vectors include viral and non-viral vectors. Among them, lipid nanoparticles (LNPs), which are lipid vesicles with a uniform lipid core, possess high biocompatibility and biodegradability, making them one of the most promising delivery vectors.

[0003] Chinese patent document CN115487306A discloses a liver-targeted drug delivery carrier, which includes a cationic polymer, a lipid complex, and a targeting molecule. The cationic polymer includes an imidazole-containing cationic polymer, the lipid complex includes an auxiliary ionized lipid molecule and a linker lipid, and the targeting molecule includes galactose-modified cholesterol-polyethylene glycol. This delivery carrier is used to deliver siRNA drugs and can improve the efficiency of siRNA drug delivery. The imidazole-containing cationic polymer can form a cationic polymer / siRNA complex with negatively charged siRNA through electrostatic interaction, which reduces non-specific interactions of the cell membrane and improves siRNA delivery and transfection efficiency.

[0004] Chinese patent document CN118267376A discloses a nucleic acid delivery vector and its preparation method and uses. The nucleic acid delivery vector is made of ionizable cationic lipids, auxiliary phospholipids, sterol compounds, lipid polyethylene glycol conjugates, and buffer solutions. Based on this, mRNA drugs or vaccines can be prepared with high efficiency, high stability and low toxicity. This nucleic acid delivery vector can achieve efficient delivery of active substances (such as mRNA).

[0005] The inventions mentioned above are all LNP delivery systems for siRNA and mRNA. However, there is little research on LNP delivery systems for DNA in the prior art, especially on delivery systems for long DNA fragments.

[0006] Circular single-stranded DNA (Css DNA) is a class of single-stranded DNA with a covalently closed topological structure, also known as single-stranded plasmids. It possesses advantages such as weak immunogenicity, low integration risk, and programmable structure. In the field of DNA nanomaterials, long Css DNA is mostly used as the basic backbone of DNA origami structures. However, research on custom-encoded long Css DNA in in vitro and in vivo systems is limited. It is well known that mRNA and Css DNA have similar yet different structures; delivery systems suitable for mRNA may not be suitable for Css DNA, and existing LNP formulations for Css DNA delivery are inefficient. Therefore, LNP delivery systems for long circular single-stranded DNA still need to be developed and explored. Summary of the Invention

[0007] This invention provides a circular single-stranded DNA delivery vector that exhibits excellent transfection efficiency when delivering circular single-stranded DNA at an N / P ratio of 6:1.

[0008] The specific technical solution adopted is as follows:

[0009] A circular single-stranded DNA delivery vector comprising an ionizable cationic lipid, a lipid polyethylene glycol conjugate, a sterol compound, and 1-stearoyl-2-oleoyl lecithin;

[0010] With the total number of moles of lipids present in the circular single-stranded DNA delivery vector as 1, the molar percentage of ionizable cationic lipids is 25%-60%, the molar percentage of lipid polyethylene glycol conjugates is 1.0%-3.5%, the molar percentage of sterol compounds is 20%-50%, and the molar percentage of 1-stearoyl-2-oleoyl lecithin is 5%-30%.

[0011] When the circular single-stranded DNA delivery vector delivers circular single-stranded DNA, the ratio of the positive and negative charges of the ionizable cationic lipids and the circular single-stranded DNA in the circular single-stranded DNA delivery vector is 4-10:1.

[0012] Preferably, the ionizable cationic lipid is SM-102, the lipid polyethylene glycol conjugate is at least one of DMG-PEG-2000, DSPE-PEG-2000, FA-DMG-PEG-2000, and FA-DSPE-PEG-2000, and the sterol compound is cholesterol;

[0013] With the total number of moles of total lipids present in the circular single-stranded DNA delivery vector as 1, the molar percentage of SM-102 is 50%, the molar percentage of lipid polyethylene glycol conjugate is 1.5%, the molar percentage of cholesterol is 38.5%, and the molar percentage of 1-stearoyl-2-oleoyl lecithin is 10%.

[0014] When the circular single-stranded DNA delivery vector delivers circular single-stranded DNA, the ratio of the positive to negative charge of the ionizable cationic lipids and the circular single-stranded DNA in the circular single-stranded DNA delivery vector is 6:1.

[0015] More preferably, the lipid polyethylene glycol conjugate is a mixture of DSPE-PEG-2000 and FA-DSPE-PEG-2000 (molar ratio of 1:1-10, more preferably 1:5-10). When the above-mentioned lipid polyethylene glycol conjugate is used, the circular single-stranded DNA delivery vector has better targeting of tumor cells.

[0016] The present invention also provides a lipid nanoparticle loaded with circular single-stranded DNA, comprising the circular single-stranded DNA delivery carrier and the circular single-stranded DNA, wherein the circular single-stranded DNA is encapsulated within the circular single-stranded DNA delivery carrier, and the ratio of positive to negative charge (N / P ratio) of the ionizable cationic lipid and the circular single-stranded DNA in the circular single-stranded DNA delivery carrier is 4-10:1, further 4-8:1, and even further 6:1.

[0017] The inventors conducted extensive research during the experiment and found that the expression of Css DNA in the Css DNA@LNP system was the best and the effect was optimal under the condition of N / P ratio of 6:1.

[0018] Preferably, the circular single-stranded DNA has ≥500 bases, more preferably ≥1000 bases, and even more preferably ≥2000 bases.

[0019] More preferably, the circular single-stranded DNA is Css IL-12, with the sequence shown in SEQ ID NO.1. Using the optimized delivery vector described above to deliver the circular single-stranded DNA, the circular single-stranded DNA can serve as a long-acting expression vector for protein substitution, enabling controlled and long-acting expression of cytokines and gene therapy.

[0020] The present invention also provides the application of the lipid nanoparticles loaded with circular single-stranded DNA in the preparation of disease treatment products, disease prevention products or imaging reagents.

[0021] The present invention also provides a disease treatment and / or disease prevention product comprising the aforementioned lipid nanoparticles loaded with circular single-stranded DNA.

[0022] Optionally, the disease treatment and / or disease prevention products may also include pharmaceutically acceptable excipients.

[0023] Optionally, the disease treatment and / or disease prevention product is an injectable.

[0024] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0025] (1) The present invention develops a delivery vector suitable for the delivery of long circular single-stranded DNA. The delivery vector, which is formulated with a specific ratio of ionizable cationic lipids, lipid polyethylene glycol conjugates, sterol compounds and 1-stearoyl-2-oleoyl lecithin, exhibits excellent transfection efficiency in the Css DNA@LNP system constructed under the condition of N / P ratio of 4-10. The transfection efficiency in 293T cells is 92.60%, which is significantly higher than the transfection efficiency (43.2%) of LNP formulations containing DSPC in the market. This is of great significance for improving the performance of gene delivery systems.

[0026] (2) The lipid nanoparticles loaded with circular single-stranded DNA prepared by the method of the present invention have good application prospects in the preparation of disease treatment products, disease prevention products or imaging reagents.

[0027] (3) Circular single-stranded DNA is delivered using the delivery vector optimized by the present invention. Circular single-stranded DNA can serve as a long-acting expression vector for protein substitution, enabling controllable long-acting expression of cytokines and gene therapy. Attached Figure Description

[0028] Figure 1 The following are the characterization results of the four lipid nanoparticles CssDNA@LNP loaded with circular single-stranded DNA prepared in Example 1, where a) is the particle size distribution map, b) is the potential statistics map, and c) is the TEM image of S-SOPC.

[0029] Figure 2 This image shows the effect of delivering Css EGFP at the cellular level using four types of lipid nanoparticles loaded with circular single-stranded DNA prepared in Example 1.

[0030] Figure 3 This is a quantitative statistical graph showing the delivery of Css EGFP at the cellular level at 72 hours using four lipid nanoparticles loaded with circular single-stranded DNA prepared in Example 1.

[0031] Figure 4 The expression of Css Luc in mice was shown by intramuscular and tail vein injection of the three lipid nanoparticles loaded with circular single-stranded DNA prepared in Example 3.

[0032] Figure 5 The expression of Css DNA@LNP prepared with SOPC as an auxiliary lipid in Example 3 in mice was shown by intramuscular injection and tail vein injection.

[0033] Figure 6The image shows the characterization of Css DNA@LNP prepared with SOPC as an auxiliary lipid in Example 4, which delivers Css IL-12 via intratumoral administration to 4T1 breast cancer cells. In the image, a) is a schematic diagram of the drug administration process, b) is the tumor volume statistics, c) is the mouse weight statistics, and d) is the mouse survival rate statistics.

[0034] Figure 7 The image shows the characterization of Css DNA@FA-LNP prepared in Example 5, which delivers CssIL-12 via tail vein targeted drug delivery to 4T1 breast cancer cells. In the image, a) is a schematic diagram of the drug delivery process, b) is the tumor volume statistics, c) is the mouse weight statistics, and d) is the mouse survival rate statistics.

[0035] In the attached figure, ns indicates no significant difference, *p<0.05, and ***p<0.001. Detailed Implementation

[0036] The present invention will be further illustrated below with reference to the embodiments and accompanying drawings. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0037] In the following examples, the circular single-stranded DNAs Css EGFP and Css Luc can be extracted according to the literature (Tang L, Tian Z, Cheng J, et al. Circular single-stranded DNA as switchable vector for gene expression in mammalian cells[J]. Nat Commun, 2023, 14(1):6665.); the sequence of Css IL-12 is shown in SEQ ID NO.1; the number of bases of Css EGFP, Css Luc, and Css IL-12 are 2001, 3231, and 3801 bases, respectively.

[0038] Example 1

[0039] The composition of the circular single-stranded DNA delivery vector in this embodiment is shown in the table below:

[0040] Table 1. Composition of the circular single-stranded DNA delivery vector

[0041]

[0042] DSPC stands for 1,2-distearyl-sn-glycerol-3-phosphocholine, DOPC stands for 1,2-dioleoyl-sn-glycerol-3-phosphocholine, SOPC stands for 1-stearoyl-2-oleoyl lecithin, and DOPE stands for 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine.

[0043] Specifically, according to the formula in the table above, SM-102, DMG-PEG-2000, cholesterol and different auxiliary lipids were dissolved in ethanol to prepare four oil phase solutions, which were stored as 10 mg / mL mother liquor.

[0044] Css EGFP expressing EGFP was added to 100 mM citrate buffer (pH = 4.0) to prepare an aqueous solution.

[0045] In preparing lipid nanoparticles loaded with circular single-stranded DNA, the N / P ratio of SM-102 and Css EGFP was maintained at 6:1. Specifically, four aqueous solutions were vortexed (3000 rpm) on a vortex mixer until the speed was uniform. Then, the oil phase solution was vertically and rapidly added to the aqueous phase solution, and vortexed for 1 min (3000 rpm). After the mixed phase was allowed to stand for 10 min to equilibrate, four lipid nanoparticles loaded with circular single-stranded DNA, Css DNA@LNP (S-DSPC, S-DOPC, S-SOPC, S-DOPE), were obtained. Subsequently, 850 μL of DPBS buffer was added to the above Css DNA@LNP system, bringing the volume to 1 mL. After equilibration for 10 min, the mixture was added to a 100K ultrafiltration tube and equilibrated for another 10 min. The mixture was then centrifuged and concentrated to an appropriate volume (3000 rpm) for later use.

[0046] The Css DNA@LNP was characterized by its particle size, potential, and surface morphology. The results are as follows: Figure 1 As shown in a)-c), the results indicate that the four Css DNA@LNPs prepared by the above method are all uniform particles with uniform and stable particle size (all in the range of 150-200 nm) and near-neutral surface potential. At the same time, the encapsulation efficiency test results show that the encapsulation efficiency of the four delivery vectors for Css EGFP is ≥90%, which is highly efficient.

[0047] Furthermore, the expression of four Css DNA@LNPs in 293T and MCF-7 cells was observed using a 10x microscope at 24h, 48h, and 72h. The results are as follows: Figure 2 As shown, 1-stearoyl-2-oleoyllecithin (SOPC) as an auxiliary lipid resulted in the best expression of Css DNA@LNP. After 72 hours, the transfection efficiency of EGFP was quantified by flow cytometry after cell collection, and the results are as follows. Figure 3 As shown, the EGFP transfection efficiency of Css DNA@LNP prepared with SOPC as an auxiliary lipid was the highest in 293T and MCF-7 cells after 72 hours, at 92.6% and 53.7%, respectively. These results demonstrate that Css DNA@LNP prepared with SOPC as an auxiliary lipid is the most effective for delivering Css EGFP.

[0048] Example 2

[0049] In this embodiment, lipid nanoparticles loaded with circular single-stranded DNA were prepared using the aqueous phase solution prepared in Example 1 and four oil phase solutions via microfluidic method. Similarly, when preparing lipid nanoparticles loaded with circular single-stranded DNA, the ratio of positive to negative charge (N / P ratio) of SM-102 and Css EGFP was maintained at 6:1.

[0050] Specifically, the microfluidic device is started, and the aqueous and oil phase solutions are mixed using the device's accompanying microfluidic chip—an interlaced herringbone mixer. The mixing process is carried out at 12 mL / min. -1 The total flow rate was maintained at a 3:1 volume ratio of aqueous phase solution to oil phase solution (v / v). The mixed system was dialyzed with phosphate-buffered saline (DPBS) at pH 7.4 for 4-10 hours at 4°C using a dialysis bag (MWCO = 15 kDa). The prepared liquid was purified to obtain Css DNA@LNP. Finally, the sample was concentrated to a suitable volume using an Amicon Ultra-4 mL MWCO = 100 K ultrafiltration tube and stored at 4°C until use.

[0051] In the process of preparing lipid nanoparticles loaded with circular single-stranded DNA using microfluidics, a chip cleaning procedure needs to be performed each time the material formulation is changed, i.e., the total volume of anhydrous ethanol injection is 2.0 mL, the flow rate ratio is 1:1, and the total flow rate is 12 mL / min.

[0052] Example 3

[0053] The difference between this embodiment and Example 1 is that: the three oil-phase solutions prepared in Example 1 (using DSPC, DOPC, and SOPC as auxiliary lipids respectively) were used, and Css Luc expressing Luc was added to 100mM citrate buffer solution (pH=4.0) to prepare an aqueous phase solution; the preparation methods and parameters of other lipid nanoparticles loaded with circular single-stranded DNA were the same as in Example 1, resulting in three types of Css DNA@LNP.

[0054] The expression of Css Luc in vivo was evaluated using different delivery vectors via intramuscular and tail vein injection. Results from intramuscular injection 12 hours later were analyzed. Figure 4Css DNA@LNP synthesized using DSPC as a lipid aid is retained in the liver via blood circulation at the injection site, with minimal uptake and weak expression. Css DNA@LNP synthesized using DOPC as a lipid aid is primarily uptaken at the injection site. Css DNA@LNP synthesized using SOPC as a lipid aid is partially uptaken at the injection site with strong expression, and a portion also enters the liver via blood circulation for expression. Quantitative characterization at the intramuscular injection site revealed that the average total expression of Css DNA@LNP synthesized using SOPC as a lipid aid in the liver was approximately 1 × 10⁻⁶. 7 The p / s ratio was higher than that of Css DNA@LNP prepared using DOPC or DSPC as auxiliary lipids. Results from tail vein injection 12 hours later showed that all three Css DNA@LNPs were mainly expressed in the liver, and based on the expression intensity and quantification results, SOPC > DOPC > DSPC.

[0055] Further testing was conducted on the performance of SOPC as an auxiliary lipid in the preparation of Css DNA@LNP. This Css DNA@LNP was used to deliver Css Luc via intramuscular and tail vein injection. The intramuscular injection group was observed to have a duration of 182 days, while the intravenous injection group was observed to have a duration of 119 days. The results indicate that ( Figure 5 During the 182-day period, the intramuscular injection group (IM group) showed consistently high expression at the injection site. In the 119-day period, the tail vein injection group (IV group) showed high expression in the liver for the first 3 days, followed by a decrease in expression. On day 14, luciferase expression in the liver region remained at a low level, while weak luciferase expression also began to appear in the lymph nodes, maintaining this level for the subsequent 119 days. This indicates that regardless of the injection method, Css DNA maintains a prolonged state of expression.

[0056] Example 4

[0057] The difference between this embodiment and Example 1 is that: the oil phase solution with SOPC as the auxiliary lipid prepared in Example 1 was used, and Css IL-12 expressing IL-12 was added to 100mM citrate buffer solution (pH=4.0) to prepare an aqueous phase solution; the preparation methods and parameters of other lipid nanoparticles loaded with circular single-stranded DNA were the same as in Example 1, and Css DNA@LNP was obtained.

[0058] The flowchart illustrating the intratumoral drug delivery process of Css DNA@LNP for breast cancer is shown below. Figure 6As shown in a), mice were injected intratumorally with this Css DNA@LNP (loaded with 5 μg Css IL-12) on days 1, 4, and 7, with PBS and the corresponding LNP vector used as controls. It was observed that after three administrations, the tumor was inhibited and showed a certain degree of therapeutic effect. By day 60, the tumor had disappeared, and the tumor-bearing mice were cured. The results are as follows... Figure 6 As shown in b), the Css DNA@LNP group exhibited good therapeutic effects within 18 days of treatment, compared to the LNP group (without Css IL-12) and the PBS control group. Throughout the treatment, the mice's body weight did not change or fluctuate significantly, indicating that the treatment did not cause significant systemic toxicity in the mice. Figure 6 c)). Continuously observe the survival time of mice and plot survival curves, such as... Figure 6 As shown in d), the CssDNA@LNP group significantly prolonged the survival time of mice, and 6 out of 8 treated mice were cured, indicating that the material has a good therapeutic effect on breast cancer by intratumoral administration.

[0059] Example 5

[0060] The only difference between this embodiment and Example 4 is that, when preparing the oil phase solution, DMG-PEG-2000 is replaced with a mixture of FA-DSPE-PEG-2000 and DSPE-PEG-2000 (the molar ratio of FA-DSPE-PEG-2000 and DSPE-PEG-2000 is 1:9). The preparation methods and parameters of other lipid nanoparticles loaded with circular single-stranded DNA are the same as in Example 4, resulting in Css DNA@FA-LNP.

[0061] The flowchart illustrating the intratumoral drug delivery process of Css DNA@FA-LNP for breast cancer is shown below. Figure 7 As shown in a), on days 1, 4, and 7, mice were intravenously injected with the same volume of PBS, FA-LNP (without Css IL-12), IL-12LNP (Example 4, without FA), and Css DNA@FA-LNP (loaded with 10 μg Css IL-12), respectively. After three administrations, tumors were inhibited and a certain degree of therapeutic effect was observed. Figure 7As shown in b), the Css DNA@FA-LNP group exhibited significant therapeutic efficacy during treatment, demonstrating a more pronounced therapeutic effect compared to the FA-LNP group, the IL-12LNP group, and the PBS control group. In the cross-sectional comparison, the IL-12LNP group, as a non-FA-targeted LNP group, also showed an inhibitory effect on tumor growth. This result indicates that even without specific FA targeting, IL-12LNPs can potentially reach tumor tissue and exert their effects through passive diffusion. Simultaneously, IL-12LNPs that fail to directly target the tumor site may be absorbed by the body through other pathways, thereby triggering a systemic immune response. However, compared to the non-targeted group, Css DNA@FA-LNP achieves a more superior therapeutic effect by actively targeting tumor cells. Figure 7 As shown in c) of the diagram, the mice's weight did not change or fluctuate significantly throughout the treatment process, indicating that the drug administration did not cause significant systemic toxicity. The survival time of the mice was continuously observed, and survival curves were plotted, as shown in... Figure 7 As shown in d), compared with the FA-LNP group, IL-12LNP and PBS control group, the Css DNA@FA-LNP group significantly prolonged the survival time of mice, indicating that the material has a good therapeutic effect on breast cancer through targeted therapy.

[0062] The embodiments described above provide a detailed explanation of the technical solutions of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, additions, or similar substitutions made within the scope of the principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A circular single-stranded DNA delivery vector, characterized by, ionizable cationic lipids, lipid polyethylene glycol conjugates, sterol compounds, and 1-stearoyl-2-oleoyl phosphatidylcholine; the ionizable cationic lipids are 25%-60% in terms of mole percentage, the lipid polyethylene glycol conjugates are 1.0%-3.5% in terms of mole percentage, the sterol compounds are 20%-50% in terms of mole percentage, and the 1-stearoyl-2-oleoyl phosphatidylcholine is 5%-30% in terms of mole percentage, based on the total number of moles of the total lipids present in the circular single-stranded DNA delivery vehicle; when the circular single-stranded DNA delivery vehicle delivers the circular single-stranded DNA, the ratio of the positive and negative charges of the ionizable cationic lipids and the circular single-stranded DNA in the circular single-stranded DNA delivery vehicle is 4-10:

1.

2. The circular single-stranded DNA delivery vector according to claim 1, wherein the ionizable cationic lipids are SM-102, the lipid polyethylene glycol conjugates are at least one of DMG-PEG-2000, DSPE-PEG-2000, FA-DMG-PEG-2000, and FA-DSPE-PEG-2000, and the sterol compounds are cholesterol; the ionizable cationic lipids are SM-102, the lipid polyethylene glycol conjugates are at least one of DMG-PEG-2000, DSPE-PEG-2000, FA-DMG-PEG-2000, and FA-DSPE-PEG-2000, and the sterol compounds are cholesterol; when the circular single-stranded DNA delivery vehicle delivers the circular single-stranded DNA, the ratio of the positive and negative charges of the ionizable cationic lipids and the circular single-stranded DNA in the circular single-stranded DNA delivery vehicle is 6:

1.

3. A lipid nanoparticle loaded with circular single-stranded DNA, characterized in that, the circular single-stranded DNA delivery vehicle and the circular single-stranded DNA, the circular single-stranded DNA is encapsulated in the circular single-stranded DNA delivery vehicle, and the ratio of the positive and negative charges of the ionizable cationic lipids and the circular single-stranded DNA in the circular single-stranded DNA delivery vehicle is 4-10:

1.

4. The lipid nanoparticle loaded with circular single-stranded DNA of claim 3, wherein, the circular single-stranded DNA has a base number of ≥500 bases.

5. The lipid nanoparticle loaded with circular single-stranded DNA of claim 3, wherein, the circular single-stranded DNA is Css IL-12, and the sequence is shown in SEQ ID NO.

1.

6. The use of the circular single-stranded DNA-loaded lipid nanoparticle according to any one of claims 3-5 in the preparation of a disease treatment product, a disease prevention product, or an imaging reagent.

7. A disease treatment and / or disease prevention product, characterized by, The circular single-stranded DNA-loaded lipid nanoparticle according to any one of claims 3-5.

8. The disease treatment and / or disease prevention product according to claim 7, characterized in that, The disease treatment and / or disease prevention product further comprises a pharmaceutically acceptable excipient.

9. The disease treatment and / or disease prevention product according to claim 7, characterized in that, The disease treatment and / or disease prevention product is an injection. The disease treatment and / or disease prevention product is an injection.

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