Calcium Binding-Mediated Sustained Release of Minocycline from Hydrophilic Multilayer Coatings Targeting Infection and Inflammation

<div><p>Infection and inflammation are common complications that seriously affect the functionality and longevity of implanted medical implants. Systemic administration of antibiotics and anti-inflammatory drugs often cannot achieve sufficient local concentration to be effective, and elicits serious side effects. Local delivery of therapeutics from drug-eluting coatings presents a promising solution. However, hydrophobic and thick coatings are commonly used to ensure sufficient drug loading and sustained release, which may limit tissue integration and tissue device communications. A calcium-mediated drug delivery mechanism was developed and characterized in this study. This novel mechanism allows controlled, sustained release of minocycline, an effective antibiotic and anti-inflammatory drug, from nanoscale thin hydrophilic polyelectrolyte multilayers for over 35 days at physiologically relevant concentrations. pH-responsive minocycline release was observed as the chelation between minocycline and Ca²⁺ is less stable at acidic pH, enabling ‘smart’ drug delivery in response to infection and/or inflammation-induced tissue acidosis. The release kinetics of minocycline can be controlled by varying initial loading, Ca²⁺ concentration, and Ca²⁺ incorporation into different layers, enabling facile development of implant coatings with versatile release kinetics. This drug delivery platform can potentially be used for releasing any drug that has high Ca²⁺ binding affinity, enabling its use in a variety of biomedical applications.</p></div

Mechanism schematic and growth of LbL assembly.

<p>(A) Schematic illustrating the mechnism of MH incorporation and LbL assembly. (B) UV absorbance of MH during DS/MH LbL assembly. (C) UV absorbance of MH during DS/MH/GA LbL assembly. *, <i>P</i><0.05 compared with (DS+Ca²⁺/MH/GA) and (DS+Ca²⁺/MH+Ca²⁺/GA) LbL films. (D) Fluorescent intensity of FITC-GA during DS/MH/GA LbL assembly. (E) Film thickness of as a function of the number of trilayers deposited on silicon substrates. Data shown are average ±STD (n = 3).</p

The anti-biofilm activity of (DS+Ca²⁺/MH+Ca²⁺/GA+Ca²⁺)₈ LbL film.

<p>(A) XTT assays to detect surviving bacteria demonstrate inactivation of bacteria in biofilms as a result of MH release from coatings. Significant anti-biofilm activity was observed in the wells coated with films containing MH. *, <i>P</i><0.05 compared with uncoated control (polystyrene); +, <i>P</i><0.05 compared with coatings without MH. Data shown are average ±STD (n = 4). (B) Fluorescent images of <i>A. baumannii</i> from ATCC and clinical isolate cultured on uncoated polystyrene, coatings with and without MH. The cells were stained with “live” SYTO 9 stain (green) and “dead” propidium iodide stain (red). Biofilm formation was eliminated on coatings with MH. Scale bar  = 10 µm.</p

Daily MH release from 6 and 8 trilayers of (DS+Ca²⁺/MH+Ca²⁺/GA+Ca²⁺).

<p>*, <i>P</i><0.05 compared with release from 6 trilayers. Data shown are average ±STD (n = 3).</p

Effect of Ca²⁺ concentration on MH release from (DS+Ca²⁺/MH+Ca²⁺/GA+Ca²⁺)₈ LbL films.

<p>Data shown are average ±STD (n = 3).</p

Effect of Ca²⁺ incorporation on MH release from (DS+Ca²⁺/MH+Ca²⁺/GA+Ca²⁺)₈ LbL films.

<p>Data shown are average ±STD (n = 3).</p

The anti-inflammatory activity of MH released from (DS+Ca²⁺/MH+Ca²⁺/GA+Ca²⁺)₈ LbL film.

<p>NO production by macrophages treated with LPS, LPS and fresh MH (1 µg/ml), or LPS and MH released on day 32 (diluted to 1 µg/ml). NO level in cells without any treatment was used as control. *, <i>P</i><0.05 compared with LPS-treated culture. Data shown are average ±STD (n = 3).</p

Long-term effect of thymectomy plus prednisone versus prednisone alone in patients with non-thymomatous myasthenia gravis: 2-year extension of the MGTX randomised trial

© 2019 Elsevier Ltd Background: The Thymectomy Trial in Non-Thymomatous Myasthenia Gravis Patients Receiving Prednisone (MGTX) showed that thymectomy combined with prednisone was superior to prednisone alone in improving clinical status as measured by the Quantitative Myasthenia Gravis (QMG) score in patients with generalised non-thymomatous myasthenia gravis at 3 years. We investigated the long-term effects of thymectomy up to 5 years on clinical status, medication requirements, and adverse events. Methods: We did a rater-blinded 2-year extension study at 36 centres in 15 countries for all patients who completed the randomised controlled MGTX and were willing to participate. MGTX patients were aged 18 to 65 years at enrolment, had generalised non-thymomatous myasthenia gravis of less than 5 years\u27 duration, had acetylcholine receptor antibody titres of 1·00 nmol/L or higher (or concentrations of 0·50–0·99 nmol/L if diagnosis was confirmed by positive edrophonium or abnormal repetitive nerve stimulation, or abnormal single fibre electromyography), had Myasthenia Gravis Foundation of America Clinical Classification Class II–IV disease, and were on optimal anticholinesterase therapy with or without oral corticosteroids. In MGTX, patients were randomly assigned (1:1) to either thymectomy plus prednisone or prednisone alone. All patients in both groups received oral prednisone at doses titrated up to 100 mg on alternate days until they achieved minimal manifestation status. The primary endpoints of the extension phase were the time-weighted means of the QMG score and alternate-day prednisone dose from month 0 to month 60. Analyses were by intention to treat. The trial is registered with ClinicalTrials.gov, number NCT00294658. It is closed to new participants, with follow-up completed. Findings: Of the 111 patients who completed the 3-year MGTX, 68 (61%) entered the extension study between Sept 1, 2009, and Aug 26, 2015 (33 in the prednisone alone group and 35 in the prednisone plus thymectomy group). 50 (74%) patients completed the 60-month assessment, 24 in the prednisone alone group and 26 in the prednisone plus thymectomy group. At 5 years, patients in the thymectomy plus prednisone group had significantly lower time-weighted mean QMG scores (5·47 [SD 3·87] vs 9·34 [5·08]; p=0·0007) and mean alternate-day prednisone doses (24 mg [SD 21] vs 48 mg [29]; p=0·0002) than did those in the prednisone alone group. 14 (42%) of 33 patients in the prednisone group, and 12 (34%) of 35 in the thymectomy plus prednisone group, had at least one adverse event by month 60. No treatment-related deaths were reported during the extension phase. Interpretation: At 5 years, thymectomy plus prednisone continues to confer benefits in patients with generalised non-thymomatous myasthenia gravis compared with prednisone alone. Although caution is appropriate when generalising our findings because of the small sample size of our study, they nevertheless provide further support for the benefits of thymectomy in patients with generalised non-thymomatous myasthenia gravis. Funding: National Institutes of Health, National Institute of Neurological Disorders and Stroke