Mixing versus Polymer Chemistry in the Synthesis of Loaded Polymer Nanoparticles through Nanoprecipitation.

Polymer nanoparticles (NPs) loaded with drugs and contrast agents have become key tools in the advancement of nanomedicine, requiring robust technologies for their synthesis. Nanoprecipitation is a particularly interesting technique for the assembly of loaded polymer NPs, which is well-known to proceed under kinetic control, with a strong influence of the assembly conditions. On the other hand, the nature of the used polymer also influences the outcome of nanoprecipitation. Here, we investigated systematically the relative effects of mixing of the organic and aqueous phases and polymer chemistry on the formation of polymer nanocarriers. For this, two mixing schemes, manual mixing and microfluidic mixing using an impact-jet micromixer, were first evaluated, showing mixing times of several tens of milliseconds and a few milliseconds, respectively. Copolymers of ethyl methacrylate with charged and hydrophilic groups and different polyesters (poly(d-l-lactide-co-glycolide) and poly(lactic acid)) were combined with a fluorescent dye salt and tested for particle assembly using these "slow" and "fast" mixing methods. Our results showed that in the case of the most hydrophobic polymers, the speed of mixing had no significant influence on the size and loading of the formed NPs. In contrast, in the case of less hydrophobic polymers, faster mixing led to smaller NPs with better encapsulation. The switch between mixing and polymer-controlled assembly was directly correlated to the solubility limit of the polymers in acetonitrile-water mixtures, with a critical point for solubility limits between 15 and 20 vol % of water. Our results provide simple guidelines on how to evaluate the possible influence of polymer chemistry and mixing on the formation of loaded NPs, opening the way to fine-tune their properties and optimize their large-scale production.

Long-Range Energy Transfer between Dye-Loaded Nanoparticles: Observation and Amplified Detection of Nucleic Acids.

Förster resonance energy transfer (FRET) is essential in optical materials for light-harvesting, photovoltaics, and biosensing, but its operating range is fundamentally limited by the Förster radius of ≈5 nm. In this work, FRET between fluorescent organic nanoparticles (NPs) is studied in order to break this limit. The donor and acceptor NPs are built from charged hydrophobic polymers loaded with cationic dyes and bulky hydrophobic counterions. Their surface is functionalized with DNA in order to control surface-to-surface distance. It is found that the FRET efficiency does not follow the canonic Förster law, reaching 0.70 and 0.45 values for NP-NP distances of 15 and 20 nm, respectively. This corresponds to the FRET efficiency decay as power four of the surface-to-surface NP-NP distance. Based on this long-distance FRET, a DNA nanoprobe is developed, where a target DNA fragment, encoding the cancer marker survivin, bringing together donor and acceptor NPs at ≈15 nm distance. In this nanoprobe, a single-molecular recognition results in unprecedented color switch for >5000 dyes, yielding a simple and fast assay with 18 attomoles limit of detection. Breaking the Förster distance limit for ultrabright NPs opens the route to advanced optical nanomaterials for amplified FRET-based biosensing.

Biotinylated Fluorescent Polymeric Nanoparticles for Enhanced Immunostaining.

The performance of fluorescence immunostaining is physically limited by the brightness of organic dyes, whereas fluorescence labeling with multiple dyes per antibody can lead to dye self-quenching. The present work reports a methodology of antibody labeling by biotinylated zwitterionic dye-loaded polymeric nanoparticles (NPs). A rationally designed hydrophobic polymer, poly(ethyl methacrylate) bearing charged, zwitterionic and biotin groups (PEMA-ZI-biotin), enables preparation of small (14 nm) and bright fluorescent biotinylated NPs loaded with large quantities of cationic rhodamine dye with bulky hydrophobic counterion (fluorinated tetraphenylborate). The biotin exposure at the particle surface is confirmed by Förster resonance energy transfer with dye-streptavidin conjugate. Single-particle microscopy validates specific binding to biotinylated surfaces, with particle brightness 21-fold higher than quantum dot-585 (QD-585) at 550 nm excitation. The nanoimmunostaining method, which couples biotinylated antibody (cetuximab) with bright biotinylated zwitterionic NPs through streptavidin, significantly improves fluorescence imaging of target epidermal growth factor receptors (EGFR) on the cell surface compared to a dye-based labeling. Importantly, cetuximab labeled with PEMA-ZI-biotin NPs can differentiate cells with distinct expression levels of EGFR cancer marker. The developed nanoprobes can greatly amplify the signal from labeled antibodies, and thus become a useful tool in the high-sensitivity detection of disease biomarkers.

Assembly of Fluorescent Polymer Nanoparticles Using Different Microfluidic Mixers.

Nanoprecipitation is a facile and efficient approach to the assembly of loaded polymer nanoparticles (NPs) for applications in bioimaging and targeted drug delivery. Their successful use in clinics requires reproducible and scalable synthesis, for which microfluidics appears as an attractive technique. However, in the case of nanoprecipitation, particle formation depends strongly on mixing. Here, we compare 5 different types of microfluidic mixers with respect to the formation and properties of poly(d-l-lactide-co-glycolide) (PLGA) and poly(methyl methacrylate) NPs loaded with a fluorescent dye salt: a cross-shaped mixer, a multilamination mixer, a split and recombine mixer, two herringbone mixers, and two impact jet mixers. Size and fluorescence properties of the NPs obtained with these mixers are evaluated. All mixers, except the cross-shaped one, yield NPs at least as small and fluorescent as those obtained manually. Notably in the case of impact jet mixers operated at high flow speeds, the size of the NPs could be strongly reduced from >50 nm down to <20 nm. Surprisingly, the fluorescence quantum yield of NPs obtained with these mixers also depends strongly on the flow speed, increasing, in the case of PLGA, from 30 to >70%. These results show the importance of precisely controlling the assembly conditions for loaded polymer NPs. The present work further provides guidance for choosing the optimal microfluidic setup for production of nanomaterials for biomedical applications.

Protein-like particles through nanoprecipitation of mixtures of polymers of opposite charge.

HYPOTHESIS: Polymer nanoparticles (NPs) have a very high potential for applications notably in the biomedical field. However, synthetic polymer NPs cannot yet concurrence the functionalities of proteins, their natural counterparts, notably in terms of size, control over internal structure and interactions with biological environments. We hypothesize that kinetic trapping of polymers bearing oppositely charged groups in NPs could bring a new level of control and allow mimicking the surfaces of proteins. EXPERIMENTS: Here, the assembly of mixed-charge polymer NPs through nanoprecipitation of mixtures of oppositely charged polymers is studied. Two series of copolymers made of ethyl methacrylate and 1 to 25 mol% of either methacrylic acid or a trimethylammonium bearing methacrylate are synthesized. These carboxylic acid or trimethylammonium bearing polymers are then mixed in different ratios and nanoprecipitated. The influence of the charge fraction, mixing ratio of the polymers, and precipitation conditions on NP size and surface charge is studied. FINDINGS: Using this approach, NPs of less than 25 nm with tunable surface charge from +40 mV to -40 mV are assembled. The resulting NPs are sensitive to pH and certain NP formulations have an isoelectric point allowing repeated charge reversal. Encapsulation of fluorescent dyes yields very bright fluorescent NPs, whose interactions with cells are studied through fluorescence microscopy. The obtained results show the potential of nanoprecipitation of oppositely charged polymers for the design of NPs with precisely tuned surface properties.

Low rate of dislocation of dual-mobility cups in primary total hip arthroplasty.

BACKGROUND: Dual-mobility (DM) cups were introduced to minimize the risk of THA dislocation. The overall rate of dislocation of DM cups (including both large and small articulations) is controversial and ranges from 0% to 5% in previous studies. QUESTIONS/PURPOSES: We therefore recorded (1) the dislocation rate, (2) loosening and osteolysis, and (3) subsequent related revisions with DM cups. METHODS: Between 1998 and 2003, 2480 primary THAs with DM cups were undertaken in 2179 patients. The mean age was 69 years (range, 19-94 years). This group underwent specific clinical and radiographic evaluation at a minimum followup of 0.17 years (mean, 7 years; range, 0.17-11 years) to assess dislocation, reoperation, osteolysis, and cup fixation. RESULTS: There were 22 dislocations (0.88%): 15 dislocations of large articulations (0.6%), with two (0.08%) recurring but only one requiring revision (0.04%), and seven intraprosthetic small articulation dislocations (0.28%), all needing revision surgery. At last followup, mean Harris hip score was 91 (range, 60-100); 2439 cups (98%) showed no signs of loosening; and 141 patients (145 hips) had osteolysis (6%). Osteolysis and cup loosening were more frequent in patients younger than 50 years at the time of surgery. The 10-year survivorship considering revision for any reason was 93% (95% CI, 91%-95%). CONCLUSIONS: DM cups had a low dislocation rate in primary THA, with a limited frequency of adverse effects. We recommend DM cups to minimize dislocation in populations at high risk for instability, but they should be avoided in younger, active patients at higher risk for osteolysis.

Recurrence of dislocation following total hip arthroplasty revision using dual mobility cups was rare in 180 hips followed over 7 years.

BACKGROUND: Dual mobility (DM) cups of mobile polyethylene were introduced to prevent total hip arthroplasty (THA) dislocation, but no large series with this design to treat recurrent instability have been reported. PURPOSE: Our retrospective investigation ascertained the efficiency of DM cups in correction of recurrent dislocation and assessed any adverse effects. METHODS: One hundred eighty THAs with recurrent instability were revised to DM cups in 180 patients (mean age, 67.4 ± 11.7 years; range, 19 to 92 years). Thirty-one patients (17.2%) underwent at least one earlier THA revision, and 15 (10.3%) incurred non-union of the greater trochanter. Of the initial group in 2009, 145 patients had completed evaluations which included assessment of the Harris Hip Score and a radiographic assessment at a mean follow-up of 7.7 ± 2.2 years (range, 4 to 14 years). The rate of survival was calculated considering any reason for revision as failure. RESULTS: At follow-up, Harris hip score was 83.9 ± 16.1 (range, 21 to 100). Dislocation of the large articulation occurred in seven hips (4.8%), and only two recurred (1.4%) (one requiring additional revision). In addition, two intra-prosthetic dislocations of the small articulation (1.4%) were observed and needed revision surgery. The large number of earlier surgeries and non-union of the greater trochanter were related to recurrent instability. Two cups (1.4%) showed signs of definite loosening; six (4.1%) presented signs of possible loosening. Twenty-nine hips manifested femoral or acetabular osteolysis (20%), but only three were severe. Eight-year survival rate considering revision for any reason was 92.6% (95% CI, 85.5-96.4%). CONCLUSIONS: This series indicates that DM cups are a viable option to treat recurrent THA instability. Their design provides a low risk of recurrent instability without increasing mechanical complications.

Metal-on-Metal Bearing: Is This the End of the Line? We Do Not Think So.

BACKGROUND: Recent studies have recommended the discontinuation of metal-on-metal (MoM) components in total hip arthroplasty (THA) because of adverse effects reported with large-diameter MoM THA. This is despite favorable long-term results observed with 28 and 32 mm MoM bearings. QUESTIONS/PURPOSES: The aim of this study was to assess the value of calls for an end to MoM bearings as THA components. Specifically, we wish to address the risks associated with MoM bearings including adverse soft tissue reactions, metal ion release, and carcinogenic risk. METHODS: The study evaluates the arguments in the literature reporting on MoM (adverse soft tissue reactions, metal ion release, and carcinogenic risk) and the experience of the current authors who re-introduced these bearings in 1995. They are balanced by a benefit-risk review of the literature and the authors' experience with MoM use. RESULTS: Adverse reactions to metallic debris as well as metal ion release are predictable and can be prevented by adequate design (arc of coverage, clearance), metallurgy (forged instead of cast alloy, high-carbide content), and appropriate component orientation. There is no scientific evidence that carcinogenicity is increased in subjects with MoM hip prostheses. MoM articulations appear to be attractive allowing safe hip resurfacing, decreasing the risk of THA revision in active patients, and providing secure THA fixation with cement in cages in severely deformed hips. MoM bearings in women of child-bearing age are controversial, but long-term data on metallic devices in adolescents undergoing spinal surgery seem reassuring. DISCUSSION: Adequate selection of MoM articulations ensures their safe use. These articulations are sensitive to orientation. Fifteen years of safe experience with 28- and 32-mm bearings of forged alloy and high-carbide content is the main reason for retaining them in primary and revision THA.

Metal-on-metal cups cemented into reinforcement rings: a possible new acetabular reconstruction procedure for young and active patients.

The purpose of this study was to evaluate the clinical and radiological results of Metasul cups cemented into reinforcement rings for young and active patients. Twenty-three total hip arthroplasties with Metasul cups were cemented into Muller reinforcement rings. Mean follow-up was 6.1 years (5-10). At final follow-up, the Harris hip score increased from 62.2 (39-85) to 95.2 (84-100, P = .01): no revision was undertaken for aseptic loosening or fixation failure. Considering reoperation and bearing revision as end points, survival rates were 95.8% and 100%, respectively. The mean blood concentrations of chromium, cobalt, and titanium were 1.85 µg/L, 1.24 µg/L, and 9.62 µg/L, respectively. A longer follow-up is mandatory, but it seems possible to use hard-on-hard bearings with metallic rings in young patients during hip revisions or in dysplastic cases with encouraging intermediate follow-up results.