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Early diagnosis of Parkinson's disease (PD) could significantly improve outcomes for patients and future disease-modifying treatments. Several studies have revealed that α-synuclein levels in peripheral erythrocytes are associated with PD, but the diagnostic value in early PD is still unknown.This study included both cross-sectional and longitudinal design. The subjects included 45 patients with early PD and 79 age-matched healthy controls. Participants were re-examined with repeated blood collection and clinical assessments after 3 years. The electrochemiluminescence assay was used to measure total and oligomeric α-synuclein levels respectively. The diagnostic value of erythrocytic α-synuclein for early PD was determined by receiver operator characteristic (ROC) curve. Correlations between RBC α-synuclein levels and changes over 3 years in clinical characteristic scores were further investigated with a linear regression.Total and oligomeric α-synuclein levels in erythrocyte were significantly increased in early PD groups compared with control group (Total α-synuclein, p 0.001; Oligomer, p 0.001). Levels of total and oligomeric α-synuclein in erythrocytes were correlated with MDS-UPDRS III scores in early PD (Total α-synuclein, p = 0.008; Oligomer, p = 0.037). After adjusting for age, gender and dopaminergic medication, an association was found between higher erythrocytic oligomeric α-synuclein levels at baseline and greater increase in MDS-UPDRS III scores over 3 years (p = 0.007).Our study suggests that total and oligomeric α-synuclein in erythrocyte were elevated even in the initial motor stage of PD. Higher erythrocytic oligomeric α-synuclein levels at baseline predicts a faster clinical decline over time in patients with early PD.

We conducted this multicenter retrospective study to evaluate the prevalence, clinical patterns, and risk factors for tigecycline-induced liver injury, which is a type of drug-induced liver injury (DILI).Inpatients receiving intravenous tigecycline for ≥7 days were included. Patient information was collected to assess possible DILIs. The pattern and severity of tigecycline DILI were evaluated. A multivariable logistic regression model was used to identify the independent risk factors associated with tigecycline DILI.A total of 986 patients were identified and 397 patients were included in this study. The prevalence of tigecycline DILI was 10.3% (95% confidence interval [CI] = 7.51-13.7%). The most common type of tigecycline DILI was cholestatic, with mild severity observed in most cases. Abnormal baseline alanine aminotransferase levels (odds ratio [OR] = 3.11, 95% CI = 1.55-6.24, P = 0.001), intensive care unit admission (OR = 2.63, 95% CI = 1.32-5.36, P = 0.006), and treatment length (in weeks) (OR = 1.25, 95% CI = 1.05-1.49, P = 0.011) were independent risk factors for tigecycline DILI.Our results indicate that the prevalence of tigecycline DILI is high, and that the patients at risk should receive special attention.

Accumulation of α-synuclein (α-syn) in neurons is a prominent feature of Parkinson's disease (PD). Recently, researchers have considered that extracellular vesicles (EVs) may play an important role in protein exportation and propagation, and α-syn-containing EVs derived from the central nervous system (CNS) have been detected in peripheral blood. However, mechanistic insights into CNS-derived EVs have not been well-described.Likely neurogenic EVs were purified from the plasma of PD patients and healthy controls using a well-established immunoprecipitation assay with anti-L1CAM-coated beads. A Prnp-SNCAPD patient-derived L1CAM-purified (likely neurogenic) EVs facilitated α-syn pathology and neuron loss in Prnp-SNCANovel_miR_44438 in PD-derived L1CAM-purified EVs inhibits the α-syn efflux from neurons thereby promoting the pathological accumulation and aggregation of α-syn. ANN NEUROL 2022;92:230-245.

ObjectiveThe objective of this study was to assess the status and trends of antiviral treatment in outpatients with herpes zoster in China.MethodsPrescription data on antiviral drugs were extracted from the database of the Hospital Prescription Analysis Program of China according to the inclusion criteria. Yearly prescriptions and costs were calculated, and trends were analyzed. The trends were further stratified by age, sex, and specific drug use. The distribution of defined daily costs (DDCs) of valaciclovir and famciclovir were analyzed, and trends in the median DDCs were identified.ResultsA total of 132,911 prescriptions from 49 hospitals located in six major areas of China were included in the analysis. The yearly prescriptions containing antivirals increased from 8,819 in 2010 to 16,361 in 2019. The percentage of prescriptions for patients aged 65 years and above also increased (27.7% in 2010 to 31.0% in 2019), and the number of prescriptions for females was higher than those for males (P < 0.001). The average cost of antivirals per prescription decreased; thus, the yearly cost showed no increasing trend. The main prescribed antivirals were valaciclovir and famciclovir, which progressively increased in prescriptions. The use of acyclovir decreased during the study period. Prescriptions containing topical formulations, acyclovir and penciclovir, both increased. The DDCs of valaciclovir and famciclovir decreased dramatically.ConclusionThe use of antivirals has increased over the decade, while the cost has not. Antiviral treatments adhere well to recent recommendations, except for the use of topical antivirals. The findings of this study may benefit the healthcare source allocation and management of herpes zoster in China.

Objectives Polymyxin B is a last-line antibiotic for multidrug-resistant gram-negative bacterial infections. However, limited safety and pharmacokinetic information is available. We investigated the safety and pharmacokinetics of intravenous polymyxin B in healthy subjects. Methods An open-label, single-dose clinical trial was conducted in healthy Chinese subjects. Polymyxin B (sulphate) was administered intravenously at 0.75 or 1.5 mg/kg (n = 10 per dose, 5 males and 5 females) to examine the safety and pharmacokinetics. Results One female subject in the 1.5-mg/kg group discontinued due to abdominal pain during administration. The most frequently reported adverse events were perioral paraesthesia, dizziness, and numbness of extremities (7/10 subjects in the 0.75-mg/kg group, all subjects in the 1.5-mg/kg group). All neurotoxicity-related events dissipated without treatment within a maximum of 23 h. Notably, abdominal pain (3/5) and vulvar pruritus (2/5), colpitis (2/5) or abnormal uterine bleeding (1/5) were reported in female subjects receiving the 1.5-mg/kg dose. In the 0.75-mg/kg group, the total clearance, volume of distribution and half-life of polymyxin B were 0.028±0.002 L/h/kg, 0.219±0.023 L/kg and 5.44±0.741 h, respectively; similar values were observed in the 1.5-mg/kg group. Urinary recovery was 3.7 ± 1.1% and 8.1 ± 1.3% in the 0.75- and 1.5-mg/kg groups, respectively. Population pharmacokinetics of polymyxin B was consistent with a three-compartment model. The clearance and distribution of the central compartment were 0.027 L/h/kg and 0.071 L/kg, respectively. Conclusions This study is the first to examine the safety and pharmacokinetics of polymyxin B in healthy subjects. Our results highlight that acute toxicity is a dose-limiting factor for intravenous polymyxin B.

Background Immune system dysfunction, including higher levels of peripheral monocytes and inflammatory cytokines, is an important feature of Parkinson’s disease (PD) pathogenesis, although the mechanism underlying the process remains to be investigated. In the central nervous system, it is well-known that α-synuclein (α-syn), a key protein involved in PD, activates microglia potently, and it is also reported that α-syn exists in the peripheral system, especially in erythrocytes or red blood cells (RBC) at exceedingly high concentration. The current study focused on the possibility that RBC-derived α-syn mediates the sensitization of peripheral monocytes in PD patients. Methods The hyperactivation of monocytes was assessed quantitatively by measuring mRNA levels of typical inflammatory cytokines (including IL-1β, IL-6 and TNF-α) and protein levels of secreted inflammatory cytokines (including pro-inflammatory cytokines: IL-1β, IL-6, TNF-α, IL-8, IFN-γ, IL-2, and IL-12p70 and anti-inflammatory cytokines: IL-4, IL-10, and IL-13). Western blot, nanoparticle tracking analysis and electron microscopy were used to characterize RBC-derived extracellular vesicles (RBC-EVs). Inhibitors of endocytosis and leucine-rich repeat kinase 2 (LRRK2), another key protein involved in PD, were used to investigate how these two factors mediated the process of monocyte sensitization by RBC-EVs. Results Increased inflammatory sensitization of monocytes was observed in PD patients and PD model mice. We found that α-syn-containing RBC-EVs isolated from PD model mice or free form oligomeric α-syn induced the inflammatory sensitization of THP-1 cells, and demonstrated that endocytosis was a requirement for this pathophysiological pathway. Furthermore, the hyperactivation of THP-1 cells induced by RBC-EVs was associated with increased LRRK2 production and kinase activity. The phenomenon of inflammatory sensitization of human monocytes and increased LRRK2 were also observed by the treatment of RBC-EVs isolated from PD patients. Conclusions Our data provided new insight into how hyperactivation of monocytes occurs in PD patients, and identified the central role played by α-syn-containing RBC-EVs in this process.

Dysautonomia is common in patients with Parkinson disease (PD) since disease early phase. Scales for Outcomes in Parkinson's disease – Autonomic (SCOPA-AUT) is a well-designed scale assessing the autonomic dysfunctions of PD patients. Our objectives were to examine the autonomic dysfunction in PD and scan without evidence of dopaminergic deficit (SWEDD) patients and to assess the correlation of autonomic dysfunctions with cerebrospinal fluid (CSF) biomarkers. An analysis of the Parkinson's Progression Markers Initiative (PPMI) data including 414 PD patients, 60 SWEDD patients, and 170 healthy controls (HCs) with baseline CSF biomarker measurements and SCOPA-AUT assessments was presented. Autonomic symptoms including gastrointestinal, urinary, cardiovascular, pupillomotor, thermoregulatory and sexual dysfunctions were assessed by SCOPA-AUT scales. Spearman correlation test was used to examine the correlations between CSF measurements and each section of SCOPA-AUT scales in HCs and subjects with PD or SWEDD. More severe autonomic dysfunctions were observed in patients with SWEDD than those with PD (P

Background: A robust and rapid method for therapeutic drug monitoring (TDM) is urgently needed for polymyxin B, which is a last-line antibiotic for multidrug-resistant gram-negative bacteria infection. Methodology: A 3-min run of LC–MS/MS method was established to determine the main components of polymyxin B (polymyxin B1 and B2) in human plasma or urine. Solid-phase extraction was employed to eliminate the matrix effect from complicated samples from patients. Results: The calibration range was 0.050–5.00 and 0.0110–0.549 μg/ml for polymyxin B1 and B2, respectively, in plasma and urine. The precision and accuracy of quality controls, matrix effect, extraction recovery and stability were all validated and satisfied with the ICH requirements. The method was successfully applied to a pharmacokinetic study in healthy subjects and TDM in patients. Conclusion: The rapid LC–MS/MS method was validated for polymyxin B in plasma and urine, and robust for TDM.

Transportation of key proteins via extracellular vesicles has been recently implicated in various neurodegenerative disorders, including Parkinson’s disease, as a new mechanism of disease spreading and a new source of biomarkers. Extracellular vesicles likely to be derived from the brain can be isolated from peripheral blood and have been reported to contain higher levels of α-synuclein (α-syn) in Parkinson’s disease patients. However, very little is known about extracellular vesicles in multiple system atrophy, a disease that, like Parkinson’s disease, involves pathological α-syn aggregation, though the process is centred around oligodendrocytes in multiple system atrophy. In this study, a novel immunocapture technology was developed to isolate blood CNPase-positive, oligodendrocyte-derived enriched microvesicles (OEMVs), followed by fluorescent nanoparticle tracking analysis and assessment of α-syn levels contained within the OEMVs. The results demonstrated that the concentrations of OEMVs were significantly lower in multiple system atrophy patients, compared to Parkinson’s disease patients and healthy control subjects. It is also noted that the population of OEMVs involved was mainly in the size range closer to that of exosomes, and that the average α-syn concentrations (per vesicle) contained in these OEMVs were not significantly different among the three groups. The phenomenon of reduced OEMVs was again observed in a transgenic mouse model of multiple system atrophy and in primary oligodendrocyte cultures, and the mechanism involved was likely related, at least in part, to an α-syn-mediated interference in the interaction between syntaxin 4 and VAMP2, leading to the dysfunction of the SNARE complex. These results suggest that reduced OEMVs could be an important mechanism related to pathological α-syn aggregation in oligodendrocytes, and the OEMVs found in peripheral blood could be further explored for their potential as multiple system atrophy biomarkers.

Background The introduction of non-VKA oral anticoagulants (NOACs) has changed the landscape of preventing thromboembolism events in many countries. However, the prescription trends of oral anticoagulant (OAC) in China are still unclear, which were evaluated in this study through data extracted and summarized from 5 major cities as representatives. Methods This study was designed as a time-series study which was based on pharmacy prescription data. Analysis was performed on yearly aggregated visits and expenditure. The results were also stratified by indications and specialties. Results A total of 189,006 prescriptions of 67 hospitals in 6 years were included in the study. The average growth rates of overall visit and expenditure of OAC were 15.8 and 57.5%, respectively. The share of warfarin decreased and NOACs had taken 92% of cost, covering 28% of patients in 2017. The more frequently used NOACs were rivaroxaban and dabigatran. The use of OAC was differed by indication and specialty. Conclusion The use of NOACs was found increasing rapidly in both visits and cost, sharing a majority of cost with a minority of patients. Attentions should be paid on the rational use of NOACs.

Haematologic side effects associated with piperacillin/tazobactam (PTZ) treatment are rare but can be fatal. The exact mechanism by which PTZ causes haemorrhages has not been determined. We report the case of a patient who received PTZ treatment and developed a coagulopathy through a vitamin K-dependent mechanism. A 70-year-old female patient was admitted to the intensive care unit because she had a severe case of pneumonia and a pulmonary thromboembolism. Empirical antibiotic treatment using PTZ was started. Her international normalised ratio (INR) was already increased and became elevated again when PTZ was restarted. Her coagulopathy was reversed by supplementation with vitamin K. We conclude that PTZ can induce coagulopathy through a vitamin K-dependent mechanism and, to our knowledge, this is the first case report to suggest that mechanism. We suggest that patients at risk for a vitamin K deficiency should be monitored for coagulopathy when piperacillin or any other board spectrum antibiotic is administered.

Alzheimer's disease is a devastating neurodegenerative disease that requires pharmacological intervention. We conducted a descriptive study using pharmacy prescription data of antidementia drugs. Prescription information of 99 541 patients was obtained from 55 hospitals. Overall the number of Alzheimer's disease outpatients of sampling days increased from 10 239 in 2012 to 20 546 in 2017. The main age range of patients suffering Alzheimer's disease was 75-84, while the patients aged above 85 was increasing. Nonusers of antidementia drugs, cholinesterase inhibitors (ChEIs) and memantine slowly decreased over the study period. The percentage of patients taking ChEIs and ChEIs-combined memantine increased. The most frequently prescribed ChEI was donepezil. These results are mostly in line with the guideline recommendations and clinical evidence during the study period. Efforts should be made to reduce the number of nonusers and optimize the use of antidementia drugs.

Background: Vernal keratoconjunctivitis (VKC) is a common eye disease and can result in permanent decrease or loss of vision. Houttuynia eye drops (HED) is used for the treatment of VKC. However, the clinical evidence of HED has not been well concluded. Herein, we described a proposed systemic review and meta-analysis to evaluate the clinical efficacy of HED for the treatment of VKC. Methods: Six electronic databases (Medline, Embase, the Cochrane database, Chinese National Knowledge Infrastructure, Wanfang database, and Chinese Biology and Medicine database) will be searched for randomized controlled trials (RCTs) which evaluating the clinical efficacy of HED for the treatment of VKC. Studies meet the eligibility criteria will be included. Data of the included studies will be extracted and the quality will also be evaluated. Data synthesis will be performed using RevMan software. Sensitivity analysis and publication bias will also be investigated. Results: This study will provide high-quality systemic review and synthesis of RCTs on efficacy of HED for the treatment of VKC. Conclusion: This systemic review and meta-analysis will conclude the efficacy of HED for the treatment of VKC. Registration: PROSPERO CRD42019124737

IntroductionProlonged infusion with β-lactam antibiotics should theoretically produce a better clinical efficacy than intermittent infusion in severe infection and infection caused by non-susceptible micro-organisms. The efficacy of prolonged infusion in severe infection has been well illustrated recently, but is still confusing in non-susceptible microbial infection. The objective of this meta-analysis is to determine the clinical effects of prolonged infusion with β-lactams for patients infected by microbes non-susceptible to the given drug.Methods and analysisLiterature searches will be performed with Medline, the Cochrane database, EMBASE database, Cumulative Index to Nursing and Allied Health Literature database, the Chinese National Knowledge Infrastructure and Wanfang database. Two reviewers will screen and select studies according to a priori defined eligibility criteria, and then the data from the included studies will be extracted. The quality will be evaluated based on a modified Jadad score and the Newcastle-Ottawa system for randomised controlled trials and observational studies, respectively. Data synthesis will be performed with Review Manager 5.3 software. Sensitivity analysis and publication bias will also be investigated.Ethics and disseminationNo ethics approval is required. The full article will be published in a peer-reviewed journal and presented at international conferences.PROSPERO registration numberCRD42018105111.

Objectives The aim of this study was to describe the prevalence of the use and prescribing patterns of anti-seizure medications (ASMs) over a six-year period, and to provide real-world evidence on medicine utilization of pediatric patients with epilepsy in China. Methods ASM prescriptions for pediatric patients written from 2013 to 2018 were extracted from the database of the Hospital Prescription Analysis Cooperative Project. Trends of ASM use were analyzed by total prescriptions, cost, age, sex, ASM class and specific ASM. Prescribing patterns of ASMs were also analyzed. Results A total of 44,675 ASM prescriptions were extracted for analysis in this study. Throughout the study period, a slight increase of ASM prescriptions was observed from 6170 in 2013 to 8211 in 2018. Children aged between 6 and 18 years, accounted for 78 % of total prescriptions every year. ASM use in boys was about 1.5 times higher than that in girls. Newer ASMs were prescribed more than older ASMs during this period. Sodium valproate was the most frequently prescribed ASM in 2013, and its use decreased in girls in 2016. Levetiracetam increased from 19.10 % in 2013 to 28.09 % in 2018 and became the most common ASM at the end of this study. Meanwhile, the use of oxcarbazepine increased from 19.31 % to 22.04 %, whereas the use of lamotrigine had declined from 18.43 % to 10.72 %. Monotherapy (66.24 %) was more frequently used than combined therapy, which included dual combination (25.80 %) and triple or more combinations (7.96 %). Conclusion There is an increased ASM prescription trend in childhood usage. Levetiracetam has replaced sodium valproate as the most frequently prescribed ASM in pediatric patients. Newer ASMs with fewer side effects and drug interactions are increasingly utilized, which is consistent with evolving recommendations by the medical community.

To evaluate the prescribing trends of glaucoma drugs in six major cities of China from 2013 to 2017.A descriptive analysis using pharmacy prescription data was conducted. Outpatient prescription data was extracted from the Hospital Prescription Analysis Cooperative Project. Prescribing patterns, trends of visits, and corresponding expenditures for glaucoma medications were analyzed.A total of 84297 ambulatory prescriptions were included in the current study. Visits by glaucoma patients increased from 13808 in 2013 to 20060 in 2017. Over the same period, the yearly expenditure for glaucoma drugs increased from 2.33 million to 3.95 million Chinese Yuan (CNY). Among all the six classes of glaucoma drugs (prostaglandin analogues, carbonic anhydrase inhibitors, α-receptor agonists, β-receptor antagonists, cholinergic agonists and fixed combinations), β-receptor antagonists were the most commonly prescribed in 2013, accounting for 34.3% of patients, but gradually decreased to 27.1% in 2017. Prostaglandin analogues became the most frequently prescribed drugs in 2017, accounting for 30.2% of the visits. Prostaglandin analogues are the most expensive and yielded a total expenditure of 2.34 million CNY in 2017, followed by carbonic anhydrase inhibitors, α-receptor agonists, β-receptor antagonists, fixed combinations, and cholinergic agonists. Combination therapy became increasingly prescribed in 2017.Glaucoma prescribing practices exhibited substantial changes over the study period. The number of glaucoma prescriptions continuously increased from 2013 to 2017, leading to increased prescription costs. These findings implied a similar trend observed in previous studies, as well as recommendations in the appropriate guidelines.

Aim: A photomedicine consisting of a core for photothermal therapy, a photosensitizer for photodynamic therapy, and a cancer-targeting moiety was fabricated to improve photosensitizer selectivity and antitumor efficiency. Materials & methods: Hyaluronic acid-decorated polydopamine nanoparticles with conjugated chlorin e6 (HA–PDA–Ce6) were synthesized and characterized. Cell uptake, phototoxicity, penetration, distribution and therapeutic effects were evaluated. Results: HA–PDA–Ce6 had high photoactivities for photodynamic therapy/photothermal therapy and was readily internalized via CD44-mediated endocytosis. Enhanced accumulation and deeper penetration into tumors were achieved by the diffusion molecular retention tumor targeting effect following peritumoral injection. In the combination therapy, HA–PDA–Ce6 displayed the highest tumor growth inhibition in HCT-116 tumor-bearing mice. Conclusion: HA–PDA–Ce6 is promising for targeted colorectal cancer therapy.

Background Meropenem exhibits time-dependent antimicrobial activity and prolonged infusion (PI) (extended infusion or continuous infusion, EI or CI) of meropenem can better achieve pharmacodynamics target when comparing with intermittent bolus (IB). However, the clinical outcomes between two groups remain inconclusive. Objective To evaluate current published literatures by meta-analysis to ascertain whether PI of meropenem can improve clinical outcomes. Methods Medline, Cochrane database and EMBASE were searched. Randomized control trails (RCT) and observational studies which compared the clinical outcomes of PI and IB groups were included and evaluated for quality. The data of studies were extracted and meta-analysis was performed using Revman 5.3 software. Results Six RCTs and 4 observation studies with relatively high quality were included in this analysis. Compared to IB group, PI group had a higher clinical success rate (odd ratio 2.10, 95% confidence interval 1.31–3.38) and a lower mortality (risk ratio 0.66, 95% confidence interval 0.50–0.88). The sensitivity analysis showed the results were stable. Conclusion PI of meropenem was associated with a higher clinical improvement rate and a lower mortality. It is recommended for patients with severe infection or infected by less sensitive microbial.

The aim of this study was to evaluate the skin permeability of anemonin, which was extracted from the Chinese herb weilingxian, and its potency of relieving the inflammation caused by rheumatoid arthritis (RA). To optimize the formulation, the solubility of anemonin in water and selected concentration of ethanol-water vehicles was determined. The effect of ethanol on the permeation of anemonin through human skin was then studied. Additionally, the influences of hydroxypropyl methylcellulose E50 (HPMC) and Carbomer 934 in different concentrations on the permeation of drug were investigated. Finally, the anti-inflammatory effect of the optimized formulation was assessed by murine model of xylene-induced ear edema. The results showed that the solubility and transdermal permeation of anemonin in ethanol-water vehicles linearly depended on the ethanol concentration. The combination of 30% ethanol and 3% Azone had a synergistic enhancement effect and was therefore selected for gel preparation. The 0.14% anemonin gel prepared with 1% HPMC exhibited the highest transdermal flux. The xylene-induced ear edema inhibitory rate of the optimized formulation was 48.85%. The results indicated that transdermal administration of anemonin is a potential modality for combating inflammation caused by RA.

Transfection efficiency of non-viral gene vectors is influenced by many factors, including chemical makeup, cellular uptake pathway and intracellular delivery. To investigate the effect of lipid saturation on transfection efficiency of polycation liposomes (PCLs), a soybean phospholipids (SPL), egg phospholipids (EPL) and hydrogenated soybean phosphatidylcholine (HSPC) series was used to prepare PCLs. Testing these PCLs in a luciferase assay indicated that with increasing saturation (SPL

Endosomal escape and nuclear entry are the two main barriers for successful non-viral gene delivery. To overcome these barriers, polyethylenimine (PEI) with a molecular weight of 800, conjugated to cholesterol (PEI 800-Chol) was synthesized to prepare polycation liposomes (PCLs). The effect of cationic polymers on transfection was investigated by pre-condensing DNA with these before using PCLs. The complexes of PCLs and protamine/DNA nanoparticles (PLPD) were introduced as efficient gene transfer vectors, and displayed obviously higher transfection efficiency (approximately 39-fold) than PCLs/DNA complexes. Kinetics of transgene expression indicated PLPD complexes could be maintained at a relatively high level over 72 h. The order of protamine addition affected the transfection of PLPD complexes. Pre-mixed and post-mixed PLPD complexes improved transfection, although the former was preferred. Distribution of FAM-labeled oligonucleotides (FAM-ODN) in cells mediated by PCLs were throughout the whole cell, while most FAM-ODN were nuclear when transfected with PLPD. These results suggest that the protonation of PEI and membrane destabilization of 1, 2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) increases the endosomal escape ability of vectors. The addition of protamine, containing nuclear localization signals, improved nuclear entry of DNA. The internalization pathways for PCLs involved multiple processes and were possibly dependent on cell lines.