Subtraction of liposome signals in cryo-EM structural determination of protein-liposome complexes

Reconstituting membrane proteins in liposomes and determining their structure is a common method for determining membrane protein structures using single-particle cryo-electron microscopy(cryo-EM).However,the strong signal of liposomes under cryo-EM imaging conditions often interferes with the structural determination of the embedded membrane proteins.Here,we propose a liposome signal subtraction method based on single-particle two-dimensional(2D)classification average images,aimed at enhancing the reconstruction resolution of membrane proteins.We analyzed the signal distribution characteristics of liposomes and proteins within the 2D classification average images of protein–liposome complexes in the frequency domain.Based on this analysis,we designed a method to subtract the liposome signals from the original particle images.After the subtraction,the accuracy of single-particle three-dimensional(3D)alignment was improved,enhancing the resolution of the final 3D reconstruction.We demonstrated this method using a PIEZO1-proteoliposome dataset by improving the resolution of the PIEZO1 protein.

Betamethasone Dipropionate Loaded in Nanoliposomes vs Conventional Betamethasone Dipropionate: Comparative Study of Permeability and Penetrability in Vitro and ex Vivo

A betamethasone dipropionate (BD) liposomal cream was developed to treat rheumatological, inflammatory, allergic diseases and psoriasis. BD is a corticosteroid, anti-inflammatory, and immunosuppressant. However, adverse effects are associated with prolonged topical use. For this reason, liposomes were loaded with BD because they offer excellent biocompatibility, bio adhesiveness, and penetrability that improve the effects caused by the conventional drug. Liposomal dispersions were prepared by emulsification using phospholipid 90 (lipid) and Tween 80 (surfactant). The particle size, polydispersity index (PDI), and zeta potential were measured using a particle analyzer. The betamethasone (BM) percentage of encapsulated active (EA) ingredient was also determined through High Performance Liquid Chromatography (HPLC). The Franz cell and tape stripping characterized these in vitro and ex vivo. Then the final formulation reached a particle size of 70.80 ± 3.31 nm, a PDI of 0.242 ± 0.038, a zeta potential of −11.68 ± 0.77 mv and encapsulate active of 83.1% ± 2.4, complying with the parameters of a nanotechnological formulation. In vitro and ex vivo studies confirmed significantly efficacy of the cream over the commercial product, through the greater penetration into the pig ear skin, resulting in an improved drug. Finally, the liposomal cream demonstrated significant potential for enhanced percutaneous absorption, attributed to its nanometric size. This innovative nanotechnology approach aims to reduce the frequency of topical applications, thereby minimizing the side effects associated with psoriasis treatment.

Overcoming neutrophil-induced immunosuppression in postoperative cancer therapy: Combined sialic acid-modified liposomes with scaffold-based vaccines

Immunotherapy is a promising approach for preventing postoperative tumor recurrence and metastasis. However, inflammatory neutrophils, recruited to the postoperative tumor site, have been shown to exacerbate tumor regeneration and limit the efficacy of cancer vaccines. Consequently, addressing postoperative immunosuppression caused by neutrophils is crucial for improving treatment outcomes. This study presents a combined chemoimmunotherapeutic strategy that employs a biocompatible macroporous scaffold-based cancer vaccine (S-CV) and a sialic acid (SA)-modified, doxorubicin (DOX)-loaded liposomal platform (DOX@SAL). The S-CV contains whole tumor lysates as antigens and imiquimod (R837, Toll-like receptor 7 activator)-loaded PLGA nanoparticles as immune adjuvants for cancer, which enhance dendritic cell activation and cytotoxic T cell proliferation upon localized implantation. When administered intravenously, DOX@SAL specifically targets and delivers drugs to activated neutrophils in vivo, mitigating neutrophil infiltration and suppressing postoperative inflammatory responses. In vivo and vitro experiments have demonstrated that S-CV plus DOX@SAL, a combined chemo-immunotherapeutic strategy, has a remarkable potential to inhibit postoperative local tumor recurrence and distant tumor progression, with minimal systemic toxicity, providing a new concept for postoperative treatment of tumors.

Lyophilization Process of Hydroxypropyl Tetrahydropyrantriol Liposomes

[Objectives]To enhance the skin permeability of hydroxypropyl tetrahydropyrantriol and provide a reference for the subsequent prevention or treatment of skin aging.[Methods]The lyophilization process of hydroxypropyl tetrahydropyrantriol liposomes was investigated using a single factor method,and a quality evaluation system was established based on the appearance,particle size,PDI,and re-dispersibility of the lyophilized samples.[Results]The lyophilization process of hydroxypropyl tetrahydropyrantriol liposomes was determined by single factor experiments.The pre-freezing period was 16 h at-80℃,the total drying time was 36 h,and the addition of 10%mannitol-sucrose was used as the lyoprotectant.[Conclusions]The product prepared by the lyophilization method exhibits a fluffy and full appearance,with minimal shrinkage and collapse.The volume remains consistent before and after lyophilization,and the re-dispersibility is satisfactory.The re-dissolution process is rapid,and the particle size and polydispersity index(PDI)remain largely unchanged before and after lyophilization.

Immunotherapeutic hydrogel for co-delivery of STAT3 siRNA liposomes and lidocaine hydrochloride for postoperative comprehensive management of NSCLC in a single application

Despite standard treatment for non-small cell lung cancer(NSCLC)being surgical resection,cancer recurrence and complications,such as induction of malignant pleural effusion(MPE)and significant postoperative pain,usually result in treatment failure.In this study,an alginate-based hybrid hydrogel(SOG)is developed that can be injected into the resection surface of the lungs during surgery.Briefly,endoplasmic reticulum-modified liposomes(MSLs)pre-loaded with the signal transducer and activator of transcription 3(STAT3)small interfering RNA and lidocaine hydrochloride are encapsulated in SOG.Once applied,MSLs strongly downregulated STAT3 expression in the tumor microenvironment,resulting in the apoptosis of lung cancer cells and polarization of tumor-associated macrophages towards the M1-like phenotype.Meanwhile,the release of lidocaine hydrochloride(LID)was beneficial for pain relief and natural killer cell activation.Our data demonstrated MSL@LID@SOG not only efficiently inhibited tumor growth but also potently improved the quality of life,including reduced MPE volume and pain relief in orthotopic NSCLC mouse models,even with a single administration.MSL@LID@SOG shows potential for comprehensive clinical management upon tumor resection in NSCLC,and may alter the treatment paradigms for other cancers.

Liposomes as versatile agents for the management of traumatic and nontraumatic central nervous system disorders:drug stability,targeting efficiency,and safety

Various nanoparticle-based drug delivery systems for the treatment of neurological disorders have been widely studied.However,their inability to cross the blood–brain barrier hampers the clinical translation of these therapeutic strategies.Liposomes are nanoparticles composed of lipid bilayers,which can effectively encapsulate drugs and improve drug delivery across the blood–brain barrier and into brain tissue through their targeting and permeability.Therefore,they can potentially treat traumatic and nontraumatic central nervous system diseases.In this review,we outlined the common properties and preparation methods of liposomes,including thin-film hydration,reverse-phase evaporation,solvent injection techniques,detergent removal methods,and microfluidics techniques.Afterwards,we comprehensively discussed the current applications of liposomes in central nervous system diseases,such as Alzheimer's disease,Parkinson's disease,Huntington's disease,amyotrophic lateral sclerosis,traumatic brain injury,spinal cord injury,and brain tumors.Most studies related to liposomes are still in the laboratory stage and have not yet entered clinical trials.Additionally,their application as drug delivery systems in clinical practice faces challenges such as drug stability,targeting efficiency,and safety.Therefore,we proposed development strategies related to liposomes to further promote their development in neurological disease research.

Selective ischemic-hemisphere targeting Ginkgolide B liposomes with improved solubility and therapeutic efficacy for cerebral ischemia-reperfusion injury

Cerebral ischemia-reperfusion injury(CI/RI)remains the main cause of disability and death in stroke patients due to lack of effective therapeutic strategies.One of the main issues related to CI/RI treatment is the presence of the blood-brain barrier(BBB),which affects the intracerebral delivery of drugs.Ginkgolide B(GB),a major bioactive component in commercially available products of Ginkgo biloba,has been shown significance in CI/RI treatment by regulating inflammatory pathways,oxidative damage,and metabolic disturbance,and seems to be a candidate for stroke recovery.However,limited by its poor hydrophilicity and lipophilicity,the development of GB preparations with good solubility,stability,and the ability to cross the BBB remains a challenge.Herein,we propose a combinatorial strategy by conjugating GB with highly lipophilic docosahexaenoic acid(DHA)to obtain a covalent complex GB-DHA,which can not only enhance the pharmacological effect of GB,but can also be encapsulated in liposomes stably.The amount of finally constructed Lipo@GB-DHA targeting to ischemic hemisphere was validated 2.2 times that of free solution in middle cerebral artery occlusion(MCAO)rats.Compared to the marketed ginkgolide injection,Lipo@GB-DHA significantly reduced infarct volume with better neurobehavioral recovery in MCAO rats after being intravenously administered both at 2 h and 6 h post-reperfusion.Low levels of reactive oxygen species(ROS)and high neuron survival in vitro was maintained via Lipo@GB-DHA treatment,while microglia in the ischemic brain were polarized from the pro-inflammatory M1 phenotype to the tissue-repairing M2 phenotype,which modulate neuroinflammatory and angiogenesis.In addition,Lipo@GB-DHA inhibited neuronal apoptosis via regulating the apoptotic pathway and maintained homeostasis by activating the autophagy pathway.Thus,transforming GB into a lipophilic complex and loading it into liposomes provides a promising nanomedicine strategy with excellent CI/RI therapeutic efficacy and industrialization prospects.

Preparation Process of Hydroxypropyl Tetrahydropyrantriol Liposomes

[Objectives] To explore the optimal process for preparing hydroxypropyl tetrahydropyrantriol liposomes. [Methods] A refractive index method was used to determine the content of hydroxypropyl tetrahydropyrantriol. Using particle size distribution and encapsulation rate as evaluation indicators, the effects of hydration time, ratio of organic phase to aqueous phase, granulation method, as well as thin film dispersion and reverse evaporation methods on liposomes preparation were investigated, and the optimal preparation method was selected. Single factor experiments were used to screen the drug phospholipid ratio, ultrasound time, and phospholipid cholesterol ratio, and the preparation process was optimized through orthogonal experiments. [Results] The optimal process of preparing hydroxypropyl tetrahydropyrantriol liposomes was as below: 1 : 10 of drug phospholipid ratio, 6 min of ultrasound time, 4 : 1 of phospholipid cholesterol ratio, (60.94%±7.24%) of entrapment efficiency, (86.44±6.08) nm of particle size, (0.195±0.077) of PDI. [Conclusions] The optimal preparation process of hydroxypropyl tetrahydropyrantriol liposomes selected by orthogonal experiment could effectively improve the encapsulation efficiency of hydroxypropyl tetrahydropyranotriol and reduce particle size. Moreover, the method was stable and reliable.

Mechanistic engineering of celastrol liposomes induces ferroptosis and apoptosis by directly targeting VDAC2 in hepatocellular carcinoma

Hepatocellular carcinoma(HCC)is one of most common and deadliest malignancies.Celastrol(Cel),a natural product derived from the Tripterygium wilfordii plant,has been extensively researched for its potential effectiveness in fighting cancer.However,its clinical application has been hindered by the unclear mechanism of action.Here,we used chemical proteomics to identify the direct targets of Cel and enhanced its targetability and antitumor capacity by developing a Cel-based liposomes in HCC.We demonstrated that Cel selectively targets the voltage-dependent anion channel 2(VDAC2).Cel directly binds to the cysteine residues of VDAC2,and induces cytochrome C release via dysregulating VDAC2-mediated mitochondrial permeability transition pore(mPTP)function.We further found that Cel induces ROS-mediated ferroptosis and apoptosis in HCC cells.Moreover,coencapsulation of Cel into alkyl glucoside-modified liposomes(AGCL)improved its antitumor efficacy and minimized its side effects.AGCL has been shown to effectively suppress the proliferation of tumor cells.In a xenograft nude mice experiment,AGCL significantly inhibited tumor growth and promoted apoptosis.Our findings reveal that Cel directly targets VDAC2 to induce mitochondria-dependent cell death,while the Cel liposomes enhance its targetability and reduces side effects.Overall,Cel shows promise as a therapeutic agent for HCC.

Leveraging immunoliposomes as nanocarriers against SARS-CoV-2 and its emerging variants

The global COVID-19 pandemic arising from SARS-CoV-2 has impacted many lives,gaining interest worldwide ever since it was first identified in December 2019.Till 2023,752 million cumulative cases and 6.8 million deaths were documented globally.COVID-19 has been rapidly evolving,affecting virus transmissibility and properties and contributing to increased disease severity.The Omicron is themost circulating variant of concern.Although success in its treatment has indicated progress in tackling the virus,limitations in delivering the current antiviral agents in battling emerging variants remain remarkable.With the latest advancements in nanotechnology for controlling infectious diseases,liposomes have the potential to counteract SARS-CoV-2 because of their ability to employ different targeting strategies,incorporating monoclonal antibodies for the active and passive targeting of infected patients.This review will present a concise summary of the possible strategies for utilizing immunoliposomes to improve current treatment against the occurrence of SARSCoV-2 and its variants.

Predicting liposome formulations by the integrated machine learning and molecular modeling approaches

Liposome is one of the most widely used carriers for drug delivery because of the great biocompatibility and biodegradability.Due to the complex formulation components and preparation process,formulation screening mostly relies on trial-and-error process with low efficiency.Here liposome formulation prediction models have been built by machine learning(ML)approaches.The important parameters of liposomes,including size,polydispersity index(PDI),zeta potential and encapsulation,are predicted individually by optimal ML algorithm,while the formulation features are also ranked to provide important guidance for formulation design.The analysis of key parameter reveals that drug molecules with logS[-3,-6],molecular complexity[500,1000]and XLogP3(≥2)are priority for preparing liposome with higher encapsulation.In addition,naproxen(NAP)and palmatine HCl(PAL)represented the insoluble and water-soluble molecules are prepared as liposome formulations to validate prediction ability.The consistency between predicted and experimental value verifies the satisfied accuracy of ML models.As the drug properties are critical for liposome particles,the molecular interactions and dynamics of NAP and PAL liposome are further investigated by coarse-grained molecular dynamics simulations.The modeling structure reveals that NAP molecules could distribute into lipid layer,while most PAL molecules aggregate in the inner aqueous phase of liposome.The completely different physical state of NAP and PAL confirms the importance of drug properties for liposome formulations.In summary,the general prediction models are built to predict liposome formulations,and the impacts of key factors are analyzed by combing ML with molecular modeling.The availability and rationality of these intelligent prediction systems have been proved in this study,which could be applied for liposome formulation development in the future.

The Applications of Mitoxantrone and Its Liposome in Adult Acute Myeloid Leukemia

Acute myeloid leukemia (AML), a rapidly progressing hematopoietic malignancy, can only be cured hopefully by hematopoietic stem cells transplantation (HSCT). Before HSCT, we usually exert effects by attempting certain regimens to induce these tumor cells to death. Administered in AML patients, the classic “3 + 7” intensive induction regimen including anthracyclines and cytarabine is recommended by guidelines worldwide. However, conventional regimens consist of anthracyclines, a category of drug limited by cumulative, dose-related, progressive myocardial damage and congestive heart failure occurs when its total doses break through the cut-off. Based on this background, mitoxantrone (MIT), an anthraquinone, was developed to a new form to reduce cardiotoxicity. Meanwhile, the nanomedicine, mitoxantrone liposome (Lipo-MIT), was characterized by improved bioavailability and limited toxicity. This drug has great therapeutic potential, but different side effects. We conclude the overall history and development of MIT and Lipo-MIT, which show controversial efficacy of MIT compared to doxorubicin and therapeutic potential of Lipo-MIT. This article reviewed the application of MIT and liposome forms in adult AML patients. .

Nonclinical Study of the Active Components of Doxorubicin Hydrochloride Liposome Injection in Vivo

Objectives: A non-clinical study was performed to establish a LC-MS/MS method to determine the in vivo active components of doxorubicin hydrochloride liposome injection in the plasma of Sprague-Dawley rats. Methods: Ten male SD rats were administered tail vein with a single dose of 10 mg/kg, and the concentrations of doxorubicin hydrochloride in plasma, heart, liver, spleen, lung, and kidney were determined by liquid chromatography-tandem mass spectrometry, and the pharmacokinetic parameters were calculated. Results: The final concentration of doxorubicin hydrochloride ranged from 500 ng/mL to 250,000 ng/mL, and the lower limit of quantification was 500 ng/mL;the main pharmacokinetic parameters: T1/2 was (19.282 ± 10.305) h, Cmax was (118514.828 ± 26155.134) ng/mL, AUC0-24 and AUC0-∞ were (1216659.205 ± 192706.268) ng/mL⋅h and (2082244.523 ± 860139.487) ng/mL⋅h, MRT0-24 and MRT0-∞ were (9.237 ± 0.423) h and (26.52 ± 14.015) h, respectively, and clearance (CL) was (0.005 ± 0.002) mL/h⋅ng. Conclusions: The method is simple, rapid, and sensitive, which can be used for the determination of doxorubicin hydrochloride concentration in the plasma of SD rats and pharmacokinetic non-clinical studies.

Microwave ablation combined with transarterial chemoembolization containing doxorubicin hydrochloride liposome for treating primary and metastatic liver cancers

Aims:To determine the safety and efficacy of microwave ablation(MWA)and transarterial chemoembolization(TACE)with doxorubicin hydrochloride liposome(DHL)in patients with primary liver cancer(PLC)and metastatic liver cancer(MLC).Materials and methods:The medical records of patients with primary or metastatic liver cancer who underwent MWA combined with TACE containing DHL from March 2019 to March 2022 were collected and analyzed.Treatment-related adverse events(AEs)were recorded.Local tumor response was evaluated according to the modified RECIST criteria.Local tumor progression-free survival(LTPFS)and overall survival(OS)were calculated using the Kaplan-Meier method.Results:Altogether,96 patients with liver cancer were included(PLC,n=45;MLC,n=51).Forty(41.7%)patients experienced AEs during treatment,and eight(8.3%)patients developed grade 3 AEs.Compared to before treatment,the serum total bilirubin level and neutrophil to lymphocyte ratio significantly increased after treatment.The median LTPFS was 14.5 months in patients with PLC and 10.7 months in patients with MLC.The median OS was not reached in patients with PLC or MLC.The 1-month and 3-month disease control rates reached more than 80%in both groups.Conclusion:MWA combined with TACE with DHL may be a safe and effective method for the treatment of liver cancer.

Liposomal dual delivery systems in visceral leishmaniasis enhance the synergistic effects of combination therapy:A promise for the future

Visceral leishmaniasis(VL)is a neglected tropical disease,and this review has summarized the current treatment scenario and its prospects.It also highlights alternative approaches used by research groups in India and around the world to develop cutting-edge and potent anti-leishmanial treatments.Even though numerous medications could be utilized to treat VL,the limitations of current treatments including their toxicity,cost,route of administration,and duration of doses,have contributed to the emergence of resistance.Combination therapy might be a better option due to its shorter duration,easier route of administration,and ability to extend the lifespan of individual drugs.However,there is a risk of not delivering both the drugs to the target site together,which can be overcome by the liposomal entrapment of those drugs and at a time knock an opportunity to reduce the dosage of amphotericin B if the combination drug provides a synergistic effect with it.Therefore,this review presents a novel strategy to fight against VL by introducing dual drug-loaded liposomes.

Immune checkpoint inhibition mediated with liposomal nanomedicine for cancer therapy

Immune checkpoint blockade(ICB)therapy for cancer has achieved great success both in clinical results and on the market.At the same time,success drives more attention from scientists to improve it.However,only a small portion of patients are responsive to this therapy,and it comes with a unique spectrum of side effects termed immunerelated adverse events(irAEs).The use of nanotechnology could improve ICBs’delivery to the tumor,assist them in penetrating deeper into tumor tissues and alleviate their irAEs.Liposomal nanomedicine has been investigated and used for decades,and is well-recognized as the most successful nano-drug delivery system.The successful combination of ICB with liposomal nanomedicine could help improve the efficacy of ICB therapy.In this review,we highlighted recent studies using liposomal nanomedicine(including new emerging exosomes and their inspired nanovesicles)in associating ICB therapy.

Synergistic chemotherapy/PTT/oxygen enrichment by multifunctional liposomal polydopamine nanoparticles for rheumatoid arthritis treatment

Amultifunctional liposomal polydopamine nanoparticle(MPM@Lipo)was designed in this study,to combine chemotherapy,photothermal therapy(PTT)and oxygen enrichment to clear hyperproliferating inflammatory cells and improve the hypoxic microenvironment for rheumatoid arthritis(RA)treatment.MPM@Lipo significantly scavenged intracellular reactive oxygen species and relieved joint hypoxia,thus contributing to the repolarization of M1 macrophages into M2 phenotype.Furthermore,MPM@Lipo could accumulate at inflammatory joints,inhibit the production of inflammatory factors,and protect cartilage in vivo,effectively alleviating RA progression in a rat adjuvant-induced arthritis model.Moreover,upon laser irradiation,MPM@Lipo can elevate the temperature to not only significantly obliterate excessively proliferating inflammatory cells but also accelerate the production of methotrexate and oxygen,resulting in excellent RA treatment effects.Overall,the use of synergistic chemotherapy/PTT/oxygen enrichment therapy to treat RA is a powerful potential strategy.

Doxorubicin Stealth Liposomes Prepared with PEG-Distearoyl Phosphatidylethanolamine and Distribution as well as Antitumor Activity in Mice

目的：研制出能够逃避体内网状内皮细胞的阿霉素隐形脂质体，并考察其在生物体内的分布以及比较阿霉素隐形脂质体与阿霉素普通脂质体的抗肿瘤活性。方法：将聚乙二醇－二硬脂酰磷脂酰乙醇胺（PEG－DSPE）同磷脂酰胆碱和胆固醇材料加在一起，用硫酸铵梯度法制备阿霉素隐形脂质体，同法制备阿霉素普通脂质体（但脂膜中不含PEG－DSPE）；通过尾静脉注射给药，比较隐形脂质体阿霉素、普通脂质体阿霉素和游离型阿霉素（盐酸阿霉素注射液）给药后在小鼠各主要脏器组织和血液中的分布情况；采用动物移植性肿瘤实验法，用H22小鼠肝癌细胞接种于小鼠右侧腋皮下形成实体瘤，考察阿霉素隐形脂质体和普通脂质体给药后对实体瘤的瘤重抑制率。结果：通过硫酸铵梯度法制备出了包封率高达95％的阿霉素隐形脂质体；同游离型阿霉素和普通脂质体阿霉素相比，隐形脂质体阿霉素在血液中浓度显著提高，循环时间显著延长，在心脏中分布的浓度显著降低；按5mg·kg^-1剂量治疗，第二天给药和第七天给药治疗方案，阿霉素隐形脂质体给药组的瘤重抑制率均显著高于普通脂质体给药组的瘤重抑制率；按10mg·kg^-1剂量治疗，隐形脂质体给药组的瘤重抑制率比普通脂质体给药组的瘤重抑制率稍高。结论：用普通脂质体给药相比，隐形脂质体阿霉素给药后延长了其在小鼠血液中的循环时间，说明经过PEG－DSPE修饰后的脂质体有逃避网状内皮细胞吞噬的功能（隐形），并且隐形脂质体阿霉素的抗肿瘤活性显著地提高。

Preparation, Stability and Immunoenhancement of APS (Astragalus polysaccharide) Liposomes

Six factors and 10 levels of each factor were selected by using the (uniform design method( with the aid of the computer for preparing APS liposomes. The optimal procedure for preparing APS liposomes was established and it can suit the large scale production in a pharmaceutical factory. The shelf-life of APS liposomes at 20℃ is 1.46 years. Diameters of the vesicles ( > 90% ) in APS liposomes are less than 1 μm, and the system is stable. At 40℃ the diameters of vesicles were not changed in three months. Pharmacological experiments revealed that APS liposomes exerted a strong immunoenhancement in mice. Studies in this paper established a foundation for the production and the clinical application of APS liposomes.

Transfection of the Human Sodium/Iodide Symporter(NIS) Gene with Liposomes and the Expression of the NIS Protein in Human Lung A549 Cancer Cells

OBJECTIVE To examine the possibility of human sodium iodide symporter （hNIS） protein expression in lung cancer cells. METHODS Human lung A549 cancer cells were thawed and cultured in vitro. The cells were divided into an experimental group transfected with a recombinant pcDNA3-hNIS plasmid and a control group transfected only with a pcDNA3 plasmid. The recombinant plasmid vector encoding the hNIS gene （pcDNA3-hNIS） was amplified, purified and identified. The hNIS gene was followed by DNA sequencing. A Western blot and an immunohistochemical assay were applied to detect the hNIS protein expression in the transfected human lung A549 cancer cells. RESULTS Restriction enzyme digestion and DNA sequencing results showed the size and direction of the inserted gene in the recombinant pcD- NA3-hNIS plasmid was correct. The Western blot method and immunohistochemical analysis showed a positive NIS protein expression in the experimental group. The NIS protein was detected mainly in the cell membranes showing a positive rate up to 70.6% with no expression of the NIS protein in the control group. There was a significant difference between two groups （P=0.000）. CONCLUSION The hNIS gene was transfected effectively into human lung A549 cancer cells mediated by Lipofectamine 2000, and was expressed with its protein in vitro.