Less radical surgery for women with early stage cervical cancer: Our experience on radical vaginal trachelectomy and laparoscopic pelvic lymphadenectomy.

We report the use of radical vaginal trachelectomy (RVT) with pelvic laparoscopic lymphadenectomy (LPL) in patients with early stages cervical cancer (FIGO stage IA2-IB1). This is a case series prospectively collected over a 6-year period (2011-2017) at the Ipswich hospital, UK. Cases were compared to a group of women with a similar stage of cervical cancer, but treated with radical hysterectomy (RH) and pelvic lymphadenectomy (PL). A total of 19 patients (group 1) underwent RVT and LPL, and 51 (group 2) had RH and PL. We included: 5/19 (26%) stage IA2 and 14/19 (74%) stage IB1. Among those, we had: 12/19 (63%) cases of squamous cell carcinoma (SCC), 7/19(37%) of adenocarcinoma. Mean hospital stay was 2.7 days (range 2-4) in group 1 versus 4.8 days (range 3-8) group 2 (p-value = 0.173). The complication rate was not statistically different between the two groups 4/19(21%) and 7/51(14%) (p-value 0.169). The mean follow-up period was 47.3 months (range 7-78) in both groups. There were no cases of recurrence in group 1 and 2/51(4%) cases in group 2, but the difference was not significant (p-value = 0.497). In our experience RVT in well-selected patients is a safe treatment option with similar oncological outcomes when compared to patients undergoing more extensive surgery for the same early stage cervical cancer.

Aurora kinase B siRNA-loaded lactoferrin nanoparticles potentiate the efficacy of temozolomide in treating glioblastoma.

AIM: To investigate the efficacy of lactoferrin nanoparticles (LfNPs) in delivering siRNA across the blood-brain barrier to treat glioblastoma multiforme (GBM) and with an additional objective of potentiation of conventional temozolomide (TMZ) chemotherapy. METHODS: Aurora kinase B (AKB) siRNA-loaded nanoparticles (AKB-LfNPs) were prepared with milk protein, lactoferrin, by water in oil emulsion method. AKB-LfNPs were tested in cell lines and in GBM orthotopic mouse model with and without TMZ treatment. RESULTS: AKB silencing, cytotoxicity and cell cycle arrest by these LfNPs were shown to be effective on GL261 cells. Tumor growth was significantly lower in AKB-LfNPs alone and in combination with TMZ treated mice and increased the survival by 2.5-times. CONCLUSION: Treatment of AKB-LfNPs to GBM mice improves life expectancy and has potential to combine with conventional chemotherapy.

Overcoming blood brain barrier with a dual purpose Temozolomide loaded Lactoferrin nanoparticles for combating glioma (SERP-17-12433).

Targeted delivery of drugs to the brain is challenging due to the restricted permeability across the blood brain barrier (BBB). Gliomas are devastating cancers and their positive treatment outcome using Temozolomide (TMZ) is limited due to its short plasma half-life, systemic toxicity and limited access through the blood-brain barrier (BBB). Nanoparticles made of Lactoferrin (Lf) protein, have been shown to enhance the pharmacological properties of drugs. Here, we report the specific ability of Lf nanoparticles to cross BBB and target over-expressed Lf receptors on glioma for enhanced TMZ delivery. TMZ-loaded Lf nanoparticles (TMZ-LfNPs) were prepared by our previously reported sol-oil method. While the Lf protein in the NP matrix aids in transcytosis across the BBB and preferential tumor cell uptake, the pH responsiveness leads to TMZ release exclusively in the tumor microenvironment. Delivery through LfNPs results in an enhanced and sustained intracellular concentration of TMZ in GL261 cells in vitro along with improving its in vivo pharmacokinetics and brain accumulation. TMZ-LfNPs treatment results in a significant reduction of tumor volume, higher tumor cell apoptosis and improved median survival in glioma bearing mice. These results demonstrate that LfNPs present an efficient TMZ delivery platform for an effective treatment of gliomas.

Photosensitizer and peptide-conjugated PAMAM dendrimer for targeted in vivo photodynamic therapy.

Challenges in photodynamic therapy (PDT) include development of efficient near infrared-sensitive photosensitizers (5,10,15,20-tetrakis(4-hydroxyphenyl)-21H,23H-porphine [PS]) and targeted delivery of PS to the tumor tissue. In this study, a dual functional dendrimer was synthesized for targeted PDT. For targeting, a poly(amidoamine) dendrimer (G4) was conjugated with a PS and a nitrilotriacetic acid (NTA) group. A peptide specific to human epidermal growth factor 2 was expressed in Escherichia coli with a His-tag and was specifically bound to the NTA group on the dendrimer. Reaction conditions were optimized to result in dendrimers with PS and the NTA at a fractional occupancy of 50% and 15%, respectively. The dendrimers were characterized by nuclear magnetic resonance, matrix-assisted laser desorption/ionization, absorbance, and fluorescence spectroscopy. Using PS fluorescence, cell uptake of these particles was confirmed by confocal microscopy and fluorescence-activated cell sorting. PS-dendrimers are more efficient than free PS in PDT-mediated cell death assays in HER2 positive cells, SK-OV-3. Similar effects were absent in HER2 negative cell line, MCF-7. Compared to free PS, the PS-dendrimers have shown significant tumor suppression in a xenograft animal tumor model. Conjugation of a PS with dendrimers and with a targeting agent has enhanced photodynamic therapeutic effects of the PS.

Targeted in vivo photodynamic therapy with epidermal growth factor receptor-specific peptide linked nanoparticles.

In targeted photodynamic therapy (tPDT), photosensitizers (PS) are targeted to disease tissue to reduce the dosage of PS and in addition to reduce the photo damage to the non-target tissue. We synthesized iron oxide nanoparticles (NP) armored with tumor targeting peptide and PS for targeted PDT. Chitosan covered Fe3O4 NPs (30 nm) were deposited with gold NPs to generate two distinct chemical surfaces. To the gold particles PS was attached with a lipoic acid linker. Human epidermal growth factor receptor (hEGFR)-specific peptide was also attached to the same particles via a nickel-nitrilotriacetic acid linker attached to the chitosan. Using these nanoparticles, peptide specific uptake and PDT mediated cell death of the SK-OV-3 cells (Her2(+) positive cells) were demonstrated by confocal microscopy, T2 imaging and viability assays. Peptide mediated preferential distribution of these NPs into tumor tissue was also shown in a xenograft tumor model. After one intravenous injection and one PDT dose, peptide bound NPs retarded tumor growth significantly compared to dark controls or treatments with NPs without peptide. The tumor retardation by targeted NPs was achieved at a PS concentration of 3.9 nmol/animal, whereas similar effect was seen with free PS at 220 nmol/animal. Therapeutic potential of these peptide containing NPs would be a useful in targeted PDT and in imaging the target tissue.

Targeting human epidermal growth factor receptor 2 by a cell-penetrating peptide-affibody bioconjugate.

Cell-penetrating peptide (CPP)-based delivery systems represent a strategy that facilitates DNA import efficiently and non-specifically into cells. To introduce specificity, we devised an approach that combines a cell-penetrating peptide, TAT-Mu (TM) and a targeting ligand, an HER2 antibody mimetic-affibody (AF), designated as TMAF to deliver nucleic acids into the cells. In this study, we synthesized TMAF protein and its truncated versions, i.e. MAF and AF, by expressing the corresponding plasmids in Escherichia coli BL21(DE3)pLysS cells. Purified TMAF binds DNA efficiently and protects plasmid DNA from DNaseI action. Transfection of HER2+ breast cancer cell lines MDA-MB-453, SK-OV-3, SK-BR-3 and an ovarian cancer cell line with plasmid DNA pCMVß-gal, resulted in enhanced ß-galactosidase activity when compared to control MDA-MB-231 cells. Maximal activity observed in MDA-MB-453 cells at DNA:TMAF:Protamine sulphate (PS) corresponding to 1:8:2 charge ratios. Further the observed gene transfection was resistant to serum, sensitive to the presence of free AF and non-toxic. Variants of TMAF although non-toxic, were far less efficient indicating the effective role of the TAT and Mu domains. The observed DNA uptake and reporter gene activity mediated by TMAFin vitro could be linked with the cell-surface density of tyrosine kinase receptor HER2 (ErbB2) levels estimated by Western blot. Further, we confirmed the efficacy of DNA transfer by TMAF protein in xenograft mouse models using MDA-MB-453 cells. Expression of ß-galactosidase as the reporter gene, upon intratumoral injection of DNA, in complex with TMAF, lends credence to specific DNA import and distribution within the tumor tissue that was attributed to high HER2 receptor overexpression in MDA-MB-453 cells. Through delivery of anti-TF hshRNA: TMAF: PS complex, we demonstrate specific knockdown of tissue factor (TF) in MDA-MB-453 cells in vitro. Most importantly, in a xenograft mouse model, we observe significant (P<0.05) and specific reduction of tumor volume when anti-TF hshRNA: TMAF: PS complex was injected intratumorally. Collectively our data indicate that AF-based chimeric peptides with nucleic acid binding properties may provide an effective tumor specific strategy to deliver therapeutic nucleic acids.

Synthesis and transfection efficiency of cationic oligopeptide lipids: role of linker.

In the design of new cationic lipids for gene transfection, the chemistry of linkers is widely investigated from the viewpoint of biodegradation and less from their contribution to the biophysical properties. We synthesized two dodecyl lipids with glutamide as the backbone and two lysines to provide the cationic headgroup. Lipid 1 differs from Lipid 2 by the presence of an amide linkage instead of an ester linkage that characterizes Lipid 2. The transfection efficiency of lipoplexes with cholesterol as colipid was found to be very high with Lipid 1 on Chinese Hamster Ovary (CHO) and HepG2 cell lines, whereas Lipid 2 has shown partial transfection efficiency on HepG2 cells. Lipid 1 was found to be stable in the presence of serum when tested in HepG2 and CHO cells albeit with lower activity. Fluorescence-based dye-binding and agarose gel-based assays indicated that Lipid 1 binds to DNA more efficiently than Lipid 2 at charge ratios of >1:1. The uptake of oligonucleotides with Lipid 1 was higher than Lipid 2 as revealed by confocal microscopy. Transmission electron microscopy (TEM) images reveal distinct formation of liposomes and lipoplexes with Lipid 1 but fragmented and unordered structures with Lipid 2. Fusion of Lipids 1 and 2 with anionic vesicles, with composition similar to plasma membrane, suggests that fusion of Lipid 2 was very rapid and unlike a fusion event, whereas the fusion kinetics of Lipid 1 vesicles was more defined. Differential scanning calorimetry (DSC) revealed a high T(m) for Lipid 1 (65.4 °C) while Lipid 2 had a T(m) of 23.5 °C. Surface area-pressure isotherms of Lipid 1 was less compressible compared to Lipid 2. However, microviscosity measured using 1,6-diphenyl-1,3,5-hexatriene (DPH) revealed identical values for vesicles made with either of the lipids. The presence of amide linker apparently resulted in stable vesicle formation, higher melting temperature, and low compressibility, while retaining the membrane fluid properties suggesting that the intermolecular hydrogen bonds of Lipid 1 yielded stable lipoplexes of high transfection efficiency.

Structural basis for the remarkable stability of Bacillus subtilis lipase (Lip A) at low pH.

Understanding the structural basis of altered properties of proteins due to changes in temperature or pH provides useful insights in designing proteins with improved stability. Here we report the basis for the pH-dependent thermostability of the Bacillus subtilis lipase (Lip A) using spectroscopic and X-ray crystallographic studies. At pH values above 7, lipase denatures and aggregates when heated at temperatures above 45 degrees C. However, at pH below 6 lipase denatures upon heating but the activity and its native structure is completely recovered upon cooling. In order to obtain the structural basis of this unusual stability of lipase, we determined high-resolution crystal structures of the lipase in two different crystal forms at pH 4.5 and 5. These structures show linear oligomerization of lipase using only two types of dimeric associations and these inter-molecular interactions are completely absent in several crystal forms of wild-type and mutant proteins obtained at basic pH. In accordance with the crystallographic studies, spectroscopic investigations reveal an invariant secondary structure in the pH range of 4-10. Quaternary organization of lipase at low pH resulted in changes in the tryptophan environment and binding of 1-anilino-8-naphthalene sulfate (ANS) at low pH. Low pH stability of the lipase is not observed in the presence of sodium chloride (>0.2 M) indicating the importance of ionic interactions at low pH. Inter- and intra-molecular ionic interactions that occur at pH below 6.0 are proposed to trap the molecule in a conformation that allows its complete refolding upon cooling.

Synthesis and in vitro evaluation of glutamide-containing cationic lipids for gene delivery.

A novel series of cationic amphiphiles based on dialkyl glutamides with cationic pyridinium head group were synthesized as potential gene delivery agents. Four cationic lipids with glutamide as linker and varying chain lengths were tested for their transfection efficiency in three cell lines. The DNA-lipid complexes were characterized for their ability to bind to DNA, protection from nuclease digestion, size, zeta-potential, and toxicity. All four lipids demonstrated efficient transfection in MCF-7, COS, and HeLa cells, and the reporter gene expression was much higher with DOPE as the helper lipid in the formulation when compared to cholesterol. Among these 14-carbon lipids, lipid 2 has shown the highest transfection efficiency, complete protection of DNA from nuclease digestion, and low toxicity. Interestingly, lipid 2 has also shown remarkable enhancement in transfection in the presence of serum.

Multiplex-PCR-based recombination as a novel high-fidelity method for directed evolution.

A new and convenient method for the in vitro recombination of single point mutations is presented. This method efficiently reduces the introduction of novel point mutations, which usually occur during recombination processes. A multiplex polymerase chain reaction (multiplex-PCR) generates gene fragments that contain preformed point mutations. These fragments are subsequently assembled into full-length genes by a recombination-PCR step. The process of multiplex-PCR-based recombination (MUPREC) does not require DNase I digestion for gene-fragmentation and is therefore easy to perform, even with small amounts of target DNA. The protocol yields high frequencies of recombination without creating a wild-type background. Furthermore, the low error rate results in high-quality variant libraries of true recombinants, thereby minimizing the screening efforts and saving time and money. The MUPREC method was used in the directed evolution of a Bacillus subtilis lipase that can catalyse the enantioselective hydrolysis of a model meso-compound. Thereby, the method was proved to be useful in producing a reliable second-generation library of true recombinants from which better performing variants were identified by using a high-throughput electrospray ionization mass spectrometry (ESI-MS) screening system.

Structural basis of selection and thermostability of laboratory evolved Bacillus subtilis lipase.

Variation in gene sequences generated by directed evolution approaches often does not assure a minimalist design for obtaining a desired property in proteins. While screening for enhanced thermostability, structural information was utilized in selecting mutations that are generated by error-prone PCR. By this approach we have increased the half-life of denaturation by 300-fold compared to the wild-type Bacillus subtilis lipase through three point mutations generated by only two cycles of error-prone PCR. At lower temperatures the activity parameters of the thermostable mutants are unaltered. High-resolution crystal structures of the mutants show subtle changes, which include stacking of tyrosine residues, peptide plane flipping and a better anchoring of the terminus, that challenge rational design and explain the structural basis for enhanced thermostability. The approach may offer an efficient and minimalist solution for the enhancement of a desired property of a protein.

Crystallization and preliminary X-ray crystallographic investigations on several thermostable forms of a Bacillus subtilis lipase.

Bacillus subtilis lipase loses activity above pH 10.5 and below pH 6.0. However, at low pH, i.e. below pH 5.0, the lipase acquires remarkable thermostability. Activity was unaltered for 2 h at 323 K at pH 4.0-5.0, although at pH values above 7.0 the activity was lost rapidly within minutes. Circular-dichroism studies indicate significant changes in the tertiary structure of the lipase, whereas the secondary-structural content remained unaltered. To elucidate the structural basis of the enhanced thermostability, three different forms have been crystallized at low pH along with three crystal forms of two thermostable mutants obtained using a directed-evolution approach.