Big impact of nanoparticles: analysis of the most cited nanopharmaceuticals and nanonutraceuticals research

a Oral and Maxillofacial Radiology, Applied Oral Sciences and Community Dental Care, Faculty of Dentistry, The University of Hong Kong, Hong Kong, China b Department of Pharmaceutical Technology, Faculty of Pharmacy, University of Coimbra, Polo das Ciências da Saúde, Azinhaga de Santa Comba, 3000-548 Coimbra, Portugal c CEB-Centre of Biological Engineering, University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal d CREA-Research Centre for Food and Nutrition, Via Ardeatina 546, 00178 Rome, Italy e Department of Pharmacy, University of Napoli Federico II, Via D. Montesano 49, 80131 Napoli, Italy f Department of Pharmacognosy, School of Pharmaceutical Sciences, Lovely Professional University, Phagwara 144411, Punjab, India g The Second Affiliated Hospital of Guizhou University of Traditional Chinese Medicine, Fei Shan Jie 32, 550003 Guiyang, China h Institute of Clinical Chemistry, University Hospital Zurich, University of Zurich, Wagistrasse 14, 8952 Schlieren, Switzerland i Institute of Genetics and Animal Biotechnology of the Polish Academy of Sciences, Jastrzebiec, 05-552 Magdalenka, Poland j Institute of Neurobiology, Bulgarian Academy of Sciences, 23 Acad. G. Bonchev str., 1113 Sofia, Bulgaria k Department of Pharmacognosy, University of Vienna, 1090 Vienna, Austria l Ludwig Boltzmann Institute for Digital Health and Patient Safety, Medical University of Vienna, Spitalgasse 23, 1090 Vienna, Austria


Introduction
According to the European Commission, nanomaterials stand for "materials which often have specific properties due to their small particle size" (European Commission, 2019). They can be described as products of nanotechnology with at least one dimension between 1 and 100 nm (De Jong and Borm, 2008). Our recent analysis of the biotechnology research literature identified nanotechnology and nanoparticles to be among the trending research themes (Yeung et al., 2019a). Due to their size-dependent properties, nanomaterials are being widely used in a range of applications offering several opportunities, but also posing inherent risks (Marques et al., 2019). The nanomaterials are regarded as chemical substances; hence they are regulated in Europe by the EU REACH (European Regulation on Registration, Evaluation, Authorization and Restriction of Chemicals).
What makes nanomaterials very interesting in both pharmaceutical and food industries is the possibility to control their properties using different types of raw materials. Several pre-requisites have to be considered upon the design of nanopharmaceuticals and nanonutraceuticals (Fig. 1), which involve the use of two main categories of organic materials: polymers and lipids. Nanopolymers are polymer molecules arranged in nanoscale to offer favorable properties, such as high biodegradability and biocompatibility, easy design, preparation and scale-up in a variety of structures with interesting bio-mimetic behaviour (Larena et al., 2008;Yang et al., 2019a;Ljubimova and Holler, 2012). These nanopolymers can be surface functionalized with targeting moieties for site-specific delivery or other useful properties. For instance, the superior properties of chitosan nanopolymers have Current Research in Biotechnology 2 (2020) 53-63 been recently reviewed in the context of serving as potential carriers for anti-cancer pharmaceuticals attributed to their biodegradability and biocompatibility (Shanmuganathan et al., 2019). Nanolipids have been put forward as an alternative carrier over polymers, particularly for lipophilic drugs, as the former use lipids existing in the human body in their composition (Souto et al., 2007), thereby reducing the risk of toxicological events (Doktorovova et al., 2016;Doktorovova et al., 2014). As lipid nanomaterials undergo similar metabolic pathways as lipids from food, they offer the opportunity to improve the bioavailability of a range of poorly soluble drugs (Muller et al., 2006;Muller et al., 2008). The nature of the compound, the lipid excipients and gastrointestinal digestion are factors to be considered in the development of these systems. Nanostructured lipid carriers (NLCs) and solid lipid nanoparticles (SLNs) represent two major types of lipid-based nanoparticles (Souto and Doktorovova, 2009;Souto and Muller, 2010). Meanwhile, inorganic nanomaterials (e.g., gold, silver, iron) are also employed in nanomedicine, for instance in cancer therapy, imaging diagnosis, drug delivery, and also to facilitate soft tissue repair (Mody et al., 2010;Dreaden et al., 2012;Mioc et al., 2019;Arisawa, 2019;Urie et al., 2018).
There are various methods for the synthesis of nanoparticles, which can be mainly classified into two large groups based on the top-down and bottom-up strategies (Fig. 2) Paliwal et al., 2014;Zahin et al., 2019). The top-down approach combines the use of some processes such as milling to create structures on a nanoscale from bigger starting materials. The bottom-up strategy creates multifaceted compounds starting from smaller materials based on synthetic processes (Biswas et al., 2012). For each strategy, the operational procedure, reaction conditions and adopted protocols can be varied, and the optimal procedures in each case are selected based on the type of material taken to start production and the desired final product (Khan et al., 2019).
Due to their remarkable properties, working with nanomaterials is nowadays considered a daily challenge for researchers (Jeevanandam et al., 2018). In the last decades, many research works dealing with nanomaterials were applied in the fields of healthcare, agriculture and foodstuff, electronics, and even cosmetics (Farokhzad and Langer, 2006). Often offering breakthrough solutions, nanomaterials widen the opportunity to exploit other administration routes of (nano)pharmaceutics (Davis et al., 2008), together with the development of innovative nanotechnologies in terms of diagnosis, imaging, and therapeutics (Petros and DeSimone, 2010). This new class of products, the nanopharmaceuticals, are being applied to improved and personalized medicines, with nanoformulation-based therapies for cancer, neurodegenerative diseases, infectious diseases, pain, and others being recently developed (Shi et al., 2017;Hasanzadeh-Kiabi, 2018;Zakharova et al., 2019;Sanchez-Lopez et al., 2019;Severino et al., 2016;Andreani et al., 2017;Jose et al., 2019).
Nanopharmaceuticals can lead to a delivery of drugs with improved physical-chemical properties i.e. solubility, pharmacokinetic enhancements, and extended half-life, in order to obtain a reduction on dose and toxicity (Weissig et al., 2014;Havel, 2016;Feng et al., 2019;Öztürk-Atar et al., 2019). When a nanopharmaceutical is developed, a broad range of parameters must be attained regarding the required characteristics of safety, efficacy, improved delivery, bioavailability, and applicability on human beings. Developments in the regulatory affairs of nanopharmaceutical to legislate correctly these goods and tightly regulate them according to the requirements for the human use are still needed. Nonetheless, they have already resulted in great changes in the pharmaceutical as well as nutraceutical industries (Abenavoli et al., 2018;Daliu et al., 2018;Daliu et al., 2019;Durazzo et al., 2019;Santini and Novellino, 2017a;Santini and Novellino, 2017b;Santini et al., 2017;. A.W.K. Yeung et al. Current Research in Biotechnology 2 (2020) 53-63 Nanotechnology applications to nutraceuticals are intensively studied in recent years, thus building up an emerging class of products: the nanonutraceuticals Pimentel-Moral et al., 2018;Watkins et al., 2015;Pimentel-Moral et al., 2019). Nutraceuticals, a portmanteau of the words 'nutrition' and 'pharmaceutical', can be defined as "the phytocomplex if they derive from a food of vegetal origin, and as the pool of the secondary metabolites if they derive from a food of animal origin, concentrated and administered in the more suitable pharmaceutical form" Santini and Novellino, 2017b). Nutraceutical applications are also intensively investigated in numerous disease areas, including cardiovascular diseases, cancer, and diabetes, among others (Banach et al., 2018;Boots et al., 2008;Braicu et al., 2017;Rossino and Casini, 2019;Yang et al., 2019b;Yeung et al., 2018a). The nanonutraceutical formulations represent respectively a valuable strategy used in managing health conditions, particularly for patients who are not eligible for a conventional pharmacological therapy. Studies on the follow up, use, and compliance of pharmaceuticals as described by recent works (Menditto et al., 2018;Menditto et al., 2015;Iolascon et al., 2016;Putignano et al., 2017), and the studies on communication strategies and assessment (Scala et al., 2016), should be referred not only to drugs but also to nutraceuticals in view of exploiting the field applicability to different health conditions. The nanotechnology could be applied for superior delivery of nutraceuticals with the aim to improve their bioavailability thereby increasing health benefits; examples of advantages of nanotechnology applied to the nutraceuticals are: efficient encapsulation and smart delivery and release from a nanoformulation. For instance, research on encapsulation of nutraceuticals into biodegradable, environment friendly nanocarriers, is ongoing to increase their absorption and the therapeutic potential. Nanonutraceuticals represent a promising challenge for the future. They should be properly assessed in order to estimate the maintenance of the respective nutraceutical properties at the nano-level, and to guarantee safety and efficacy. Follow-up studies to evaluate possible unwanted side effects are very important for both nanopharmaceutical and nanonutraceutical formulations (Wiwanitkit, 2012;Helal et al., 2019;Jones et al., 2019).
To gain insides on the overall high-impact research landscape of nanopharmaceuticals and nanonutraceuticals, this work identifies and analyzes the top 100 most cited original research articles of the outlined research area. Consequently, the overall aim of this report is to provide an overview of the nanopharmaceuticals and nanonutraceuticals research with a focus on the most important scientific outputs, as indicated by academic citations performance.

Literature search
In November 2019, a search was conducted through the Web of Science (WoS) Core Collection electronic database (Clarivate Analytics, Philadelphia, USA) to identify the nanopharmaceuticals and nanonutraceuticals publications. The following search strings were used: (1) TOPIC = ("nanopharma*" OR "nanomedic*" OR "nanodrug*" OR "nano-pharma*" OR "nano-medic*" OR "nano-drug*" OR "nano pharma*" OR "nano medic*" OR "nano drug*" OR "nanonutraceutic*" OR "nano-nutraceutic*" OR "nano nutraceutic*"); (2) TOPIC = ("nanoparticle* OR "nano-particle*" OR "nano particle*") AND TOPIC = (medic* OR pharma* OR drug* OR nutraceutic*); finally, (1) OR (2). This search strategy identified publications that mentioned the relevant words or their derivatives in the title, abstract, or keywords. We limited the search to original research articles only. The final search yielded 90,248 original articles, and they were sorted by descending order of citations. The articles were independently screened for relevance by two authors (AY and AGA). A list of top 100 most cited nanopharmaceuticals and nanonutraceuticals articles was compiled. All of the top 100 articles were written in English.

Data extraction and analysis
The bibliographic data of the screened 100 most cited articles were recorded, such as the publication year, authorship, institutions, countries/regions, journal title, publication count, and citation count. The "Analyze" and "Create Citation Report" functions of the WoS platform were utilized for the basic analyses. The "full records and cited references" were exported to VOSviewer software (version 1.6.11, www.vosviewer.com) for further bibliometric analyses. The VOSviewer software analyzes the terms used in titles and abstracts (of the top 100 most cited articles), links them to the bibliographic data, and visualizes the results by the means of a term map (Van Eck and Waltman, 2009). In a term map, the bubble size reflects how frequently a term is mentioned in the articles (multiple mentions in one article were counted once). The bubble color reflects the average citations (citations per article, CPA) of an article mentioning the term. The distance between two bubbles reflects how frequently two terms were co-mentioned among the 100 articles. Only words that appears in multiple articles (n = 2) were analyzed and visualized. The frequencies of author keywords were also analyzed by VOSviewer.
In addition, the collaboration networks of institutions and countries were analyzed by VOSviewer. Each collaboration was counted and weighed equally. The bubble size represents the number of articles. The distance between two bubbles represents how frequently the two institutions or countries collaborated. Please refer to the respective figure legends for the meaning of the bubble color.

Overall results
The top 100 most cited nanopharmaceuticals and nanonutraceuticals articles are listed in Table 1 (Table 2).

Institutions
The majority of the most prolific institutions were based in the United States. When institutions with at least 2 articles were considered, eleven institutions formed the largest collaboration network. MIT was in the center of the network, having collaborations with local partners and international partners, such as Gwangju Institute of Science and Technology (South Korea) and University of Paris -XI (France) (Fig. 4). It should be noted that University of California Los Angeles had 5 contributions to the top 100 articles but was not in the network as it collaborated with other partners instead of the schools in the University of California system.

Countries
As expected, the United States (62%) and China (12%) were the two most prolific countries. Interestingly, in this list the articles contributed by the United States and China had similar citations per article, without the high citation bias towards the former as observed in the literature of the common nutraceuticals such as curcumin  and resveratrol (Yeung et al., 2019c). The rest of the contributing countries are all from Asia and Europe. Countries with 3 contributions included the United Kingdom, France, Ireland, Japan and Netherlands. India and Singapore each had 2 contributions. Countries with 1 contribution each included Austria, Croatia, Italy, Norway, Russia, Spain, Sweden and Switzerland. These figures showed a different distribution as observed from the top 100 articles of nutraceuticals and functional foods, in which the United States topped the list with a smaller ratio (30%), the European countries had larger contributions (e.g., United Kingdom: 11%; Belgium and Finland: 8% each) and China played a smaller role (4%) (Yeung et al., 2018b). It was also different from a nanoscience literature analysis published in 2007 that found China only accounted for 1.73% of the top 1% of highly cited papers (Guan and Ma, 2007). Perhaps these data imply that recent papers contributed by China have gained much more citations than those published in the past.
Meanwhile, for the 20 countries that contributed to the top 100 articles, 11 of them formed an international collaboration network (Fig. 5). The United States collaborated with 8 countries (Norway, Switzerland, Netherlands, China, Russia, Germany, South Korea and France), China collaborated with the United States and Germany, whereas South Korea collaborated with the United States and France. These countries tended to publish the most cited articles more recently than the United Kingdom, and were involved in collaborations between the Western and Asian countries.

Journals
The top 100 articles were published in 39 journals, with the most prolific journals having high impact factors in the range of 9.580-43.070 (Table 2). There was no single journal leading others by a large number of articles. Among the top 10 most prolific journals, 3 were dedicated to nanoparticles research, namely ACS Nano, Nature Nanotechnology, and Nano Letters. Others were with focus on chemistry, materials science or multidisciplinary sciences. When WoS journal categories were considered, the leading categories were chemistry multidisciplinary (39%), materials science multidisciplinary (34%), chemistry physical (30%), nanoscience nanotechnology (28%), physics applied (17%), physics condensed matter (17%), multidisciplinary sciences (16%), and pharmacology pharmacy (9%). The summation of the percentages exceeded 100% because some journals belonged to multiple categories.

Lack of clinical trials in the top 100 list
As mentioned in the Introduction section, application of nanoparticles to cancer or tumor therapy and site-specific drug delivery have been important topics. The current results supported these notions. In particular, the top 2 articles were dealing with cancer imaging, targeting and photothermal therapy (Huang et al., 2006;Gao et al., 2004). Meanwhile, the articles concerning drug delivery in the top 20 of the list were dealing with the effects of shape, size and structures of nanoparticles on biodistribution and drug delivery (Chithrani and Chan, 2007;Geng et al., 2007;Horcajada et al., 2010;Liong et al., 2008). However, readers should be aware that there was only a single human clinical trial among the top 100 articles, namely the one conducted by Gradishar et al. (Gradishar et al., 2005) that showed a greater efficacy in slowing down tumor progression in patients with metastatic breast cancer and showed favorable safety profile of albumin-bound paclitaxel synthesized in nanoparticle size, relative to the standard size. The number of nanomedicine clinical trials is very small compared to non-nanomedicine, it  A.W.K. Yeung et al. Current Research in Biotechnology 2 (2020) 53-63 was estimated that there were 1430 nanomedicine trials published from 2005 to 2014, equivalent to approximately 0.8% of the number for nonnanomedicine (Woodson and Rodriguez, 2019). Among the nanomedicine trials, cancer was most prevalent (19.3%), particularly breast cancer (6.9%), whereas other diseases seemed to be much less prevalent (Woodson and Rodriguez, 2019). The comparably small number of clinical trials concerning nanoparticles is consistent to the situation for a popular nutraceutical -curcumin, for which 3.8% of the relevant literature were clinical trials ; and also for the ethnopharmacology literature, in which 1.3% were clinical studies (Yeung et al., 2019d). We hope that more clinical trials for nanopharmaceuticals and nanonutraceuticals will be conducted in the near future, and they will gain high citations as a recognition of the efforts.
Here, we would also like to draw readers' attention to the KeyWords Plus feature of WoS, which are "words or phrases that frequently appear in the titles of an article's references, but do not appear in the title of the article it-self… based upon a special algorithm" (https://support.clarivate.com/ ScientificandAcademicResearch/s/article/KeyWords-Plus-generationcreation-and-changes?language=en_US). In other words, KeyWords Plus are additional keywords added by WoS to the indexed articles. In the top 100 list, 3 articles had keywords related to clinical trials listed in the KeyWords Plus, namely "clinical-trial" for (Cabral et al., 2011), which was an in vivo study; "phase-I" for (Nasongkla et al., 2006), which was an in vitro study; and both "phase-I" and "clinical-trial" for (Paciotti et al., 2004), which was in vivo study. Meanwhile, the phase III trial mentioned above was tagged with "in-vivo" in the KeyWords Plus. These findings show that KeyWords Plus data should be used with caution for bibliometric purposes. Meanwhile, readers should be aware of some other limitations of this study. For example, some papers may not be indexed by Web of Science and thus not identified in this study. Since different databases count the number of citations differently, it was not possible to use multiple databases. Besides, citation count is dynamic, meaning that the top 100 list will be composed of different papers in the future.

Conclusion
This bibliometric study identified the top 100 most cited original articles about nanopharmaceuticals and nanonutraceuticals research. Over 60% of the 100 articles were published in the 2000s. The articles were cited 576-3665 times, with 20.1-261.8 citations per year. The majority of the most prolific institutions were based in the United States. Besides  the United States, China, South Korea, Canada and Germany also contributed heavily to the 100 articles. Some popular themes included drug delivery, tumor, toxicity/biocompatibility and biodistribution. Regarding materials, gold, silver and polymeric nanoparticles were the most commonly used.   6. Term map showing words appeared in at least 2 of the 100 articles. Bubble size reflects how frequently a term is mentioned in the articles (multiple mentions in one article were counted once). The bubble color reflects the average citations (citations per article, CPA) of an article mentioning the term. The distance between two bubbles reflects how frequently two terms were co-mentioned among the 100 articles. The lines illustrate the 1000 most commonly co-mentioned term pairs.  Fig. 7. Keyword map showing all author keywords that appeared in the 100 articles. Bubble size reflects how frequently a keyword is mentioned in the articles. The bubble color reflects the average citations (citations per article, CPA) of an article mentioning the keyword. The distance between two bubbles reflects how frequently two keywords were co-mentioned among the 100 articles. The lines illustrate the 1000 most commonly co-mentioned keyword pairs.